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Colchicine; b. Taxol; c. Cytochalasin. Syncytium and coenocyte. In some organisms, karyokinesis is not followed by cytokinesis as a result of which multinucleate ...
Life Sciences Fundamentals and Practice Sixth edition

Pranav Kumar | Usha Mina

I

Life Sciences Fundamentals and Practice I Sixth edition

Pranav Kumar Former faculty, Department of Biotechnology Jamia Millia Islamia (JMI), New Delhi, India

Usha Mina Associate Professor, School of Environmental Sciences, Jawaharlal Nehru University (JNU), New Delhi, India

Pathfinder Publication New Delhi, India

Pranav Kumar Former faculty, Department of Biotechnology, Jamia Millia Islamia (JMI), New Delhi, India

Usha Mina Associate Professor, School of Environmental Sciences, Jawaharlal Nehru University (JNU), New Delhi, India Life Sciences : Fundamentals and Practice Sixth edition

ISBN: 978-81-906427-0-5 (paperback) Copyright © 2017 by Pathfinder Publication, all rights reserved. This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reproduced by any mechanical, photographic, or electronic process, or in the form of a phonographic recording, nor it may be stored in a retrieval system, transmitted, or otherwise copied for public or private use, without written permission from the publisher. Publisher : Pathfinder Publication Production editor : Ajay Kumar Copy editor : Jomesh Joseph Illustration and layout : Pradeep Verma Cover design : Monu Marketing director : Arun Kumar Production coordinator : Murari Kumar Singh

Pathfinder Publication A unit of Pathfinder Academy Private Limited, New Delhi, India.

pathfinderpublication.in

iii

Preface Life Sciences have always been a fundamental area of science. The exponential increase in the quantity of scientific information and the rate, at which new discoveries are made, require very elaborate, interdisciplinary and up-to-date information and their understanding. This sixth edition of Life sciences, Fundamentals and practice includes extensive revisions of the previous edition. We have attempted to provide an extraordinarily large amount of information from the enormous and ever-growing field in an easily retrievable form. It is written in clear and concise language to enhance self-motivation and strategic learning skill of the students and empowering them with a mechanism to measure and analyze their abilities and the confidence of winning. We have given equal importance to text and illustrations. The sixth edition has a number of new figures to enhance understanding. At the same time, we avoid excess detail, which can obscure the main point of the figure. We have retained the design elements that have evolved through the previous editions to make the book easier to read. Sincere efforts have been made to support textual clarifications and explanations with the help of flow charts, figures and tables to make learning easy and convincing. The chapters have been supplemented with self-tests and questions so as to check one’s own level of understanding. Although the chapters of this book can be read independently of one another, they are arranged in a logical sequence. Each page is carefully laid out to place related text, figures and tables near one another, minimizing the need for page turning while reading a topic. We have given equal importance to text and illustrations as well. We hope you will find this book interesting, relevant and challenging. Acknowledgements Our students were the original inspiration for the first edition of this book, and we remain continually grateful to them, because we learn from them how to think about the life sciences, and how to communicate knowledge in most meaningful way. We thank, Dr. Diwakar Kumar Singh and Mr. Ajay Kumar, reviewers of this book, whose comments and suggestions were invaluable in improving the text. Any book of this kind requires meticulous and painstaking efforts by all its contributors. Several diligent and hardworking minds have come together to bring out this book in this complete form. We are much beholden to each of them and especially to Dr. Neeraj Tiwari. This book is a team effort, and producing it would be impossible without the outstanding people of Pathfinder Publication. It was a pleasure to work with many other dedicated and creative people of Pathfinder Publication during the production of this book, especially Pradeep Verma.

Pranav Kumar Usha Mina

v

Contents Chapter

1

Biomolecules and Catalysis 1.1

1.2

Amino acids and Proteins

1

1.1.1

Optical properties

3

1.1.2

Absolute configuration

1.1.3

Standard and non-standard amino acids

1.1.4

Titration of amino acids

7

1.1.5

Peptide and polypeptide

12

1.1.6

Peptide bond

1.1.7

Protein structure

1.1.8

Denaturation of proteins

1.1.9

Solubilities of proteins

1.1.10

Simple and conjugated proteins

4 5

13 16

Fibrous and globular proteins 1.2.1

Collagen

1.2.2

Elastin

1.2.3

Keratins

1.2.4

Myoglobin

1.2.5

Hemoglobin, Hb

20 21 22

22

23 24 25 25

Oxygen transport

27 28

Functional differences between Mb and Hb Factors affecting the affinity of Hb for oxygen 1.2.6 1.3

Models for the behavior of allosteric proteins

Protein folding

33

1.3.1

Molecular chaperones

1.3.2

Amyloid

1.3.3

Ubiquitin mediated protein degradation

1.3.4

N-end rule

35

38

1.4

Protein sequencing and assays

1.5

Nucleic acids

1.6

34

47

1.5.1

Nucleotides

1.5.2

Chargaff’s rules

Structure of dsDNA 1.6.1

B-DNA

53

1.6.2

Z-DNA

54

47

53

51

39

36

30 30 31

vi 1.6.3

Triplex DNA

55

1.6.4

G-quadruplex

1.6.5

Stability of the dsDNA helix

1.6.6

DNA denaturation

1.6.7

Quantification of nucleic acids

1.6.8

Supercoiled forms of DNA

56

Linking number

1.7

1.8

1.9

1.10

57

57

59

60

1.6.9

DNA: A genetic material

RNA

63

1.7.1

Alkali-catalyzed cleavage of RNA

1.7.2

RNA World hypothesis

1.7.3

RNA as genetic material

Carbohydrates

59

61

64

65 65

66

1.8.1

Monosaccharide

1.8.2

Epimers

1.8.3

Cyclic forms

1.8.4

Derivatives of monosaccharide

1.8.5

Disaccharides and glycosidic bond

1.8.6

Polysaccharides

1.8.7

Glycoproteins

1.8.8

Reducing and non-reducing sugar

Lipids

76

1.9.1

Fatty acids

1.9.2

Triacylglycerol and Wax

1.9.3

Phospholipids

1.9.4

Glycolipids

1.9.5

Steroid

1.9.6

Eicosanoid

1.9.7

Plasma lipoproteins

Vitamins 1.10.1

66

68 68

75

77 78

79 81

82 84

84 Water-soluble vitamins

85

Thiamine (Vitamin B1)

85

Riboflavin (Vitamin B2) 86

Biotin

86

Pantothenic acid Folic acid

85

87

87

Cobalamin (Vitamin B12) Pyridoxine (Vitamin B6) Ascorbic acid (Vitamin C) 1.10.2

71

73

82

Niacin

70

Fat-soluble vitamins Vitamin A (Retinol)

89 89

87 88 88

76

vii Vitamin D

89

Vitamin K

90

Vitamin E

90

1.11

Reactive oxygen species and antioxidant

1.12

Enzymes

91

92

1.12.1

Naming and classification of enzyme

1.12.2

How enzymes operate?

1.12.3

Catalytic strategies

1.12.4

Enzyme kinetics

1.12.5

Enzyme inhibition

1.12.6

Regulatory enzymes

1.12.7

Isozymes

110

1.12.8

Zymogen

110

1.12.9

Ribozyme

111

94

96

97 104 107

1.12.10 Examples of enzymatic reactions

Chapter

93

111

2

Bioenergetics and Metabolism 2.1

Bioenergetics

119

2.2

Metabolism

124

2.3

Respiration

125

2.3.1

Aerobic respiration

125

2.3.2

Glycolysis

2.3.3

Pyruvate oxidation

2.3.4

Krebs cycle

2.3.5

Anaplerotic reaction

2.3.6

Oxidative phosphorylation

2.3.7

Inhibitors of electron transport

2.3.8

Electrochemical proton gradient

2.3.9

Chemiosmotic theory

2.3.10

ATP synthase

2.3.11

Uncoupling agents and ionophores

2.3.12

ATP-ADP exchange across the inner mitochondrial membrane

2.3.13

Shuttle systems

2.3.14

P/O ratio

2.3.15

Fermentation

150

2.3.16

Pasteur effect

152

2.3.17

Warburg effect

2.3.18

Respiratory quotient

126 131

133 136 136 141 142

143

144

147

149

152

2.4

Glyoxylate cycle

2.5

Pentose phosphate pathway

152

153 154

146 146

viii 2.6

Entner-Doudoroff pathway

2.7

Photosynthesis

2.8

2.9

2.10

2.11

2.12

156

157

2.7.1

Photosynthetic pigment

2.7.2

Absorption and action spectra

2.7.3

Fate of light energy absorbed by photosynthetic pigments

2.7.4

Concept of photosynthetic unit

2.7.5

Hill reaction

2.7.6

Oxygenic and anoxygenic photosynthesis

2.7.7

Concept of pigment system

2.7.8

Stages of photosynthesis

2.7.9

Light reactions

2.7.10

Prokaryotic photosynthesis

2.7.11

Non-chlorophyll based photosynthesis

2.7.12

Dark reaction: Carbon reduction and fixation cycle

2.7.13

Starch and sucrose synthesis

Photorespiration

181

2.8.1

C4 cycle

182

2.8.2

CAM pathway

161

164

164

165 167

174 176

180

184 187

2.9.1

Gluconeogenesis

187

2.9.2

Glycogen metabolism

192

197

2.10.1

Synthesis and storage of triacylglycerols

2.10.2

Biosynthesis of fatty acid

2.10.3

Fatty acid oxidation

2.10.4

Biosynthesis of cholesterol

2.10.5

Steroid hormones and Bile acids

Amino acid metabolism

199

202 210 211

213

2.11.1

Amino acid synthesis

2.11.2

Amino acid catabolism

2.11.3

Molecules derived from amino acids

Nucleotide metabolism

213 216 221

222

2.12.1

Nucleotide synthesis

2.12.2

Nucleotide degradation

Chapter

164

168

Carbohydrate metabolism

Lipid metabolism

157

222 229

3

Cell Structure and Functions 3.1

What is a Cell?

235

3.2

Structure of eukaryotic cells

236

3.2.1

Plasma membrane

236

3.2.2

ABO blood group

3.2.3

Transport across plasma membrane

244 246

197

176

162

ix 3.3

Membrane potential

3.4

Transport of macromolecules across plasma membrane

3.5

3.4.1

Endocytosis

3.4.2

Fate of receptor

3.4.3

Exocytosis

Ribosome 3.5.1

3.6

3.7

253 263

263 268

268

269

Protein targeting and translocation

Endoplasmic reticulum

271

272

3.6.1

Endomembrane system

3.6.2

Transport of proteins across the ER membrane

3.6.3

Transport of proteins from ER to cis Golgi

Golgi complex

276

281

283

3.7.1

Transport of proteins through cisternae

3.7.2

Transport of proteins from the TGN to lysosomes

3.8

Vesicle fusion

3.9

Lysosome

3.10

Vacuoles

3.11

Mitochondria

3.12

Plastids

3.13

Peroxisome

3.14

Nucleus

3.15

Cytoskeleton

277

286

287 289 290

293 294

295 299

3.15.1

Microtubules

299

3.15.2

Kinesins and Dyneins

3.15.3

Cilia and Flagella

3.15.4

Centriole

3.15.5

Actin filament

3.15.6

Myosin

3.15.7

Muscle contraction

3.15.8

Intermediate filaments

302

303

305 306

307 309

3.16

Cell junctions

3.17

Cell adhesion molecules

3.18

Extracellular matrix of animals

3.19

Plant cell wall

320

3.20

Cell signaling

322

312

314 317 318

3.20.1

Signal molecules

323

3.20.2

Receptors

3.20.3

GPCR and G-proteins

3.20.4

Ion channel-linked receptors

3.20.5

Enzyme-linked receptors

3.20.6

Nitric oxide

3.20.7

Two-component signaling systems

323 325 334

334

341 342

284 285

x 3.20.8

Chemotaxis in bacteria

3.20.9

Quorum sensing

3.20.10 Scatchard plot 3.21

3.22

Cell Cycle

343

344 345

347

3.21.1

Role of Rb protein in cell cycle regulation

3.21.2

Role of p53 protein in cell cycle regulation

3.21.3

Replicative senescence

Mechanics of cell division

360

Mitosis

360

3.22.2

Meiosis

367

3.22.3

Nondisjunction and aneuploidy

Apoptosis

3.24

Cancer

371

374 377

Molecular basis of cancer Proto-oncogenes

379

380

Tumor suppressor genes Carcinogen

382

383

Retinoblastoma

384

Oncovirus or tumor virus Retroviral oncogenes

Chapter

358

360

3.22.1

3.23

356

385

385

4

Prokaryotes and Viruses 4.1

General features of Prokaryotes

4.2

Phylogenetic overview

4.3

Structure of bacterial cell Staining

393

Cell Wall

395

Outer membrane Glycocalyx

393

398 399

399

Surface appendages Endospores

392

397

Plasma membrane Cytoplasm

391

400

402

4.4

Bacterial genome: Bacterial chromosome and plasmid

4.5

Bacterial nutrition

4.6

403

408

4.5.1

Culture media

4.5.2

Bacterial growth

409 410

Horizontal gene transfer and genetic recombination 4.6.1

Transformation

4.6.2

Transduction

414 416

413

xi 4.6.3

Conjugation

420

4.7

Bacterial taxonomy

4.8

General features of important bacterial groups

4.9

Archaebacteria

429

4.10

Bacterial toxins

430

4.11

Control of microbial growth

4.12

Virus

4.13

425

432

436

4.12.1

Bacteriophage (Bacterial virus)

4.12.2

Life cycle of bacteriophage

4.12.3

Plaque assay

4.12.4

Genetic analysis of phage

4.12.5

Animal viruses

4.12.6

Plant viruses

Prions and Viroid 4.13.1

Chapter

426

438

439

442 445

448 458

458

Bacterial and viral disease

460

5

Immunology 5.1

Innate immunity

5.2

Adaptive immunity

5.3

Cells of the immune system

5.4

463 466 468

5.3.1

Lymphoid progenitor

5.3.2

Myeloid progenitor

468 470

Organs involved in the adaptive immune response 5.4.1

Primary lymphoid organs

5.4.2

Secondary lymphoid organs/tissues

5.5

Antigens

5.6

Major-histocompatibility complex

5.7

5.8

471

471 472

473 477

5.6.1

MHC molecules and antigen presentation

5.6.2

Antigen processing and presentation

5.6.3

Laboratory mice

479

480

482

Immunoglobulins: Structure and function

483

5.7.1

Basic structure of antibody molecule

5.7.2

Different classes of immunoglobulin

5.7.3

Action of antibody

5.7.4

Antigenic determinants on immunoglobulins

486

488

B-cell maturation and activation Clonal selection theory

483

490

494

5.9

Kinetics of the antibody response

5.10

Monoclonal antibodies and Hybridoma technology 5.10.1

488

495

Engineered monoclonal antibodies

498

497

xii 5.11

Organization and expression of Ig genes Mechanism of DNA rearrangements Allelic exclusion

504

Class switching

504

5.12

Generation of antibody diversity

5.13

T-cells and CMI

510

Thymic selection

512

505

Superantigens

517

5.14

Cytokines

5.15

The complement system

5.16

Hypersensitivity

5.17

Autoimmunity

5.18

Transplantation

5.19

Immunodeficiency diseases

5.20

Failures of host defense mechanisms

5.21

Vaccines

Chapter

502

507

T-cell maturation

5.13.1

499

518 521

525 527 528 528 529

531

6

Diversity of Life 6.1

Taxonomy

537

6.1.1

Nomenclature

537

6.1.2

Classification

6.1.3

Biological species concept

6.1.4

Phenetic and phylogenetic principles of classification

538

6.2

The five-kingdom system

6.3

Protists

6.4

6.5

545

547

6.3.1

Protozoan protists

6.3.2

Photosynthetic protists

6.3.3

Slime mold

549

6.3.4

Oomycetes

550

Fungi

550

6.4.1

Mycorrhiza

6.4.2

Lichens

Plantae

538

547 548

552

552

553

6.5.1

Plant life cycle

553

6.5.2

Algae

6.5.3

Life cycle of land plants

6.5.4

Bryophytes

6.5.5

Pteridophytes

560

6.5.6

Gymnosperm

561

6.5.7

Angiosperms

562

555

558

557

539

xiii 6.6

Animalia

566

6.7

Animal’s classification

574

6.7.1

Phylum Porifera (Pore bearing animals)

6.7.2

Phylum Cnidaria (Coelenterata)

6.7.3

Phylum Platyhelminthes (Flatworms)

6.7.4

Phylum Aschelminthes (Roundworms)

6.7.5

Phylum Annelida

577

6.7.6

Phylum Mollusca

577

6.7.7

Phylum Arthropoda

6.7.8

Phylum Echinodermata

6.7.9

Phylum Hemichordata

6.7.10

Phylum Chordata

Answers of self test Index

588

587

578

579

578 579

574

574 575 575

Chapter

01

Biomolecules and Catalysis A biomolecule is a carbon-based organic compound that is produced by a living organism. More than 25 naturally occurring chemical elements are found in biomolecules, but these biomolecules consist primarily of carbon, hydrogen, nitrogen, oxygen, phosphorus and sulfur. In terms of the percentage of the total number of atoms, four elements such as hydrogen, oxygen, nitrogen and carbon together make up over 99% of the mass of most cells. Biomolecules include both small as well as large molecules. The small biomolecules are low molecular weight (less than 1000) compound which include sugars, fatty acids, amino acids, nucleotides, vitamins, hormones, neurotransmitters, primary and secondary metabolites. Sugars, fatty acids, amino acids and nucleotides constitute the four major families of small biomolecules in cells. Large biomolecules which have high molecular weight are called macromolecules and mostly are polymers of small biomolecules. These macromolecules are proteins, carbohydrates and nucleic acids. Small biomolecules

Macromolecules

Sugars

Polysaccharides

Amino acids

Polypeptides (proteins)

Nucleotides

Nucleic acids

Fatty acids Nucleic acids and proteins are informational macromolecules. Proteins are polymers of amino acids and constitute the largest fraction (besides water) of cells. The nucleic acids, DNA and RNA, are polymers of nucleotides. They store, transmit, and translate genetic information. The polysaccharides, polymers of simple sugars, have two major functions. They serve as energy-yielding fuel stores and as extracellular structural elements.

1.1

Amino acids and Proteins

Amino acids are compounds containing carbon, hydrogen, oxygen and nitrogen. They serve as monomers (building blocks) of proteins and are composed of an amino group, a carboxyl group, a hydrogen atom, and a distinctive side chain, all bonded to a carbon atom, the α-carbon. In an α-amino acid, the amino and carboxylate groups are attached to the same carbon atom, which is called the α-carbon. The various α-amino acids differ with respect to the side chain (R group) attached to their α-carbon. The general structure of an amino acid is: a-carboxyl group

COO a-amino group

+

H3N



Ca

H

R Side chain

Figure 1.1

General structure of an amino acid.

This page intentionally left blank.

Biomolecules and Catalysis 1.1.1

3

Optical properties

All amino acids except glycine are optically active i.e. they rotate the plane of plane polarized light. Optically active molecules contain chiral carbon. A tetrahedral carbon atom with four different constituents are said to be chiral. All amino acids except glycine have chiral carbon and hence they are optically active. An optically active compound can rotate the plane of polarized light either clockwise (to the right) or counterclockwise (to the left). Optically active compounds that rotate the plane of polarized light clockwise are said to be dextrorotatory. By convention, this direction is designated by a plus sign (+). Optically active compounds that rotate the plane of polarized light counterclockwise are said to be levorotatory. This is designated by a minus sign (–). The + and – forms have also been termed d- and l-, respectively. COO +

H3N Achiral carbon



Ca

COO +

H

H

Glycine Figure 1.4

H3N



Ca

H

CH3

Chiral carbon

Alanine

Amino acids showing achiral and chiral carbon.

Optical activity is measured by polarimeter. Optical activity is the ability of an optically active compound to rotate the plane of linearly polarized light. Optical rotation is a quantitative measure of the rotation of light caused by the compound. The magnitude of optical rotation indicates the extent to which plane of linearly polarized light is rotated and sign represents the direction of rotation. Optical rotation of an optically active compound depends on the concentration of the compound, temperature, wavelength of light used, solvent used to dissolve the sample and light pathlength. The optical rotation of a solution at a given temperature and wavelength is given by Å = [α]Tλ × C × l where,

Å = observed rotation in degrees C = concentration of the solution in g/ml

l = light path length in decimeters (dm) [α]Tλ

= the specific rotation of compound at temperature, T (in degrees Celsius) and wavelength, λ (in nm). If the wavelength of the light used is 589 nm, the symbol ‘D’ is used, [α]DT.

Specific rotation is the reference value of optical rotation for a given concentration of compound at a given

temperature and fixed wavelength. At a given temperature and for a given wavelength of light, the specific rotation is defined as the observed value of optical rotation when plane polarized light is passed through a sample with a path length of 1 decimeter and a sample concentration of 1g per milliliter.

Sample tube containing a chiral compound

Normal light

Figure 1.5

Polarizer

Plane-polarized light

Rotation of plane-polarized light

When plane polarized light is passed through a solution that contains an optically active compound, there

is net rotation of the plane polarized light. The light is rotated either clockwise (dextrorotatory) or counterclockwise (levorotatory) by an angle that depends on the molecular structure and concentration of the compound, the path length and the wavelength of the light.

This page intentionally left blank.

Biomolecules and Catalysis

7

Nonstandard amino acids Although hundreds of different amino acids present in cells but only 22 different amino acids participate in protein synthesis and are incorporated ribosomically into proteins. Such amino acids are called proteinogenic or standard amino acids. Apart from the 22 standard amino acids, all other amino acids are not ribosomically incorporated into proteins are called non-standard. In addition to the standard amino acids, some proteins may contain nonstandard amino acid residues formed by post-translational modification of standard amino acid residues already incorporated into a polypeptide. These modifications are often essential for the function or regulation of a protein. Examples of some of these amino acids are 4-Hydroxyproline (derivative of proline), 5-Hydroxylysine (derivative of lysine), desmosine (derivative of lysine), N-acetylserine, N-formylmethionine and γ-carboxyglutamate (found in the blood clotting protein prothrombin). Besides their role in proteins, amino acids and their derivatives have many other biologically important functions. Many nonstandard amino acids are not found in proteins. These amino acids often occur as intermediates in the metabolic pathways for standard amino acids. For example, ornithine and citrulline are key intermediates in the biosynthesis of arginine and in the urea cycle. Similarly, azaserine, a nonstandard amino acid, acts as an antibiotic. It was originally thought that all unconventional amino acids were made by modifying one of the standard amino acids after it was incorporated into protein, a process called a post-translational modification. But amino acids like selenocysteine, pyrrolysine are inserted into proteins by the translational machinery. Selenocysteine (Sec or U) is the 21st standard amino acid. It has a structure similar to that of cysteine, but it contains

selenium rather than sulphur. It is incorporated into polypeptides during translation. However, it is specified by a triplet codon, UGA (a stop codon). Selenocysteine has its own tRNA containing the anticodon UCA and it is formed by modifying a serine that has been attached to the selenocysteine tRNA. Enzymes like glutathione peroxidase and formate dehydrogenase contain selenocysteine in their catalytic center. Pyrrolysine (Pyl or O) is the 22nd standard amino acid. It is similar to lysine and is present in some bacterial proteins. It is coded by UAG codon. COO



+

H3N

C

H

CH2 CH2 CH2 COO



+

H3N

COO



CH2

+

C

H

CH2 SH

H3N

C

H

NH

CH2 Se

C=O H3C N

H Cysteine

1.1.4

Selenocysteine

Pyrrolysine

Titration of amino acids

Because amino acids contain ionizable groups, the predominant ionic form of these molecules in solution depends on the pH. Titration of an amino acid illustrates the effect of pH on amino acid structure. Consider alanine, a simple amino acid, which has two titrable groups (α-amino and α-carboxyl group). During titration with a strong base such as NaOH, alanine loses two protons in a stepwise fashion. In a strongly acidic solution, alanine is present mainly in the form in which the carboxyl group is uncharged. Under this condition the molecule’s net charge is +1, since the ammonium group is protonated. However, an increase in the pH results in the deprotonation of α-carboxyl group.

8

Biomolecules and Catalysis

At this point, alanine has no net charge and is electrically neutral. The pH at which this occurs is called the isoelectric point (pI).

+

H3N

+1

0

COOH

COO pk1

Ca

H

–1 —

COO pk2

+

Ca

H3N

CH3

H

H2N

CH3

Low pH (pH < pI)



Ca

H

CH3 High pH (pH > pI)

Intermediate pH (pH = pI)

Because there is no net charge at the isoelectric point, amino acids are electrophoretically non-mobile and least soluble at this pH. Further increase in pH i.e. lowering of the H+ concentration results in the deprotonation of the charged amino group and an uncharged amino group forms. So at high pH, the net charge on the molecule is –1, since the ammonium group is deprotonated and a net negative charge develops due to the presence of the carboxylate group. 13 Alanine pK2 = 9.7

7 pH

pI = 6

pK1 = 2.34

0

0.5

1

1.5

2



OH (equivalents)

Figure 1.6

Titration curve of alanine (monoamino and monocarboxylic acid). A plot of the dependence of the pH on

the amount of OH– added is called a titration curve.

The isoelectric point for alanine may be calculated as follows: pI =

pK1 + pK2 2

The pK1 and pK2 values for alanine are 2.34 and 9.7 respectively. The pI value for alanine is therefore, pI =

2.34 + 9.7 = 6.0 2

Amino acids with ionizable side chains have more complex titration curves. Glutamic acid, for example, has a carboxyl side chain group. At low pH, glutamic acid has net charge +1. As the base is added (pH increases), the α-carboxyl group loses a proton to become a carboxylate group (in a polyprotic acid, the protons are first lost from the group with the lowest pKa). Glutamate now has no net charge. As still more base is added, the second carboxyl group

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Biomolecules and Catalysis

13

peptides are cyclic in nature. Two cyclic decapeptides (peptides containing 10 amino acid residues) produced by the bacterium Bacillus brevis are common examples. Both of these peptides, gramicidin S and tyrocidine A, are antibiotics, and both contain D-amino acids as well as L-amino acids. In addition, both contain the amino acid ornithine, which does not occur in proteins. Small peptides play many roles in organisms. Some, such as oxytocin and vasopressin, are important hormones. Others, like glutathione, regulate oxidation–reduction reactions. Still others, such as enkephalins, are naturally occurring painkillers. Aspartame is a commercially synthesized dipeptide, L-aspartylphenylalanyl methylester, and is used as an artificial sweetener. When many amino acid residues are joined, the product is called a polypeptide. Amino acids which have been incorporated into a peptide or polypeptide are termed amino acid residues. By convention, in a polypeptide the left end represented by the first amino acid while the right end represented by the last amino acid. The first amino acid is also called as N-terminal amino acid residue. The last amino acid is called the C-terminal amino acid residue.

N-terminal

H2N

H

O

C

C

R

H

O

N

C

C

H

R

Amino acid residue

Figure 1.10

H

O

N

C

C

H

R

Amino acid residue

C-terminal

OH

Amino acid residue

A series of amino acids joined by peptide bonds form a polypeptide chain, and each amino acid unit in a

polypeptide is called a residue. A polypeptide chain has polarity because its ends are different, with an α-amino group at one end and an α-carboxyl group at the other.

The peptide bonds in proteins are formed between the α-amino and the α-carboxyl groups. But peptides do occur naturally where the peptide linkage involves a carboxyl or amino group which is attached to a carbon atom other than the α-carbon. For example a dipeptide formed between the γ-carboxyl group of glutamic acid and the amino group of alanine is called γ-glutamylalanine.

1.1.6

Peptide bond

Peptides and polypeptides are linear and unbranched polymers composed of amino acids linked together by peptide bonds. Peptide bonds are amide linkages formed between α-amino group of one amino acid and the α-carboxyl group of another. This reaction is a dehydration reaction, that is, a water molecule is removed and the linked amino acids are referred to as amino acid residues. Peptide bond formation is an endergonic process, with ΔG ~ +21kJ/mol.

H2N

H

O

C

C

H

OH

R1

H

O

N

C

C

H

R2

OH

H2O

H2N

H

O

C

C

R1

H

O

N

C

C

H

R2

OH

Figure 1.11 The formation of a peptide bond (also called an amide bond) between the α-carboxyl group of one amino acid to the α-amino group of another amino acid is accompanied by the loss of a water molecule.

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16

Biomolecules and Catalysis

1.1.7

Protein structure

Proteins are unbranched polymers constructed from 22 standard α-amino acids. They have four levels of the structural organization. Primary structure, the amino acid sequence, is specified by genetic information. As the polypeptide chain folds, it forms certain localized arrangements of adjacent amino acids that constitute secondary structure. The overall three-dimensional shape that a polypeptide assumes is called the tertiary structure. Proteins that consist of two or more polypeptide chains (or subunits) are said to have a quaternary structure.

Primary structure The primary structure (1° structure) of a polypeptide is its amino acid sequence. The amino acids are connected by peptide bonds. Primary structure of polypeptide determines the higher levels of structural organization.

Secondary structure The most common types of secondary structure (2° structure) are the α-helix and the β-pleated sheet. Both α-helix and β-pleated sheet patterns are stabilized by hydrogen bonds between the carbonyl and N—H groups in the polypeptide’s backbone.

α-helix The α-helix is a rigid, rod like structure that forms when a polypeptide chain twists into a helical conformation. The screw sense of α-helix can be right-handed (clockwise) or left-handed (counterclockwise). However, right-handed helices are energetically more favorable. In almost all proteins, the helical twist of the α-helix is right-handed. There are 3.6 amino acid residues per turn of the helix and the pitch (the distance between corresponding points per turn) is 0.54 nm. Each residue is related to the next one by a rise of 1.5 Å (0.15 nm) along the helix axis. A single turn of α-helix involves 13 atoms from O to the H of the H bond. For this reason, the α-helix is referred to as the 3.613-helix. Length of α-helix is usually 10–15 amino acid residues. Intrachain hydrogen bonds form between the N—H group of each amino acid and the carbonyl group of the amino acid four residues away.

H

O1

O

C Ca

2

3

N

Ca 4

5

C

N 6

n

Figure 1.16

7

Ca

C 8

9

N

Ca

O

N

C

10

n+5

11

C

O

12

Ca

Ca N

n+4

n+2

H

H 13 n+3

n+1

H

O

H

The hydrogen bonding arrangement in the α-helix. The α-helix is known as the 3.613-helix, where 3.6

is the number of residues per turn and 13 is the number of atoms in the hydrogen-bonded loop.

Except for amino acids near the ends of an α-helix, all the main-chain CO and NH groups are hydrogen bonded. The side chains of amino acids extend outward from the helix. All H bonds lie parallel to the helix axis and point in the same direction. Helices can be formed from either D- or L-amino acids, but a given helix must be composed entirely of amino acids of one configuration. The α-helix cannot be formed from a mixed copolymer of D- and L-amino acids. L-amino acids can form either right or left handed α-helices.

Biomolecules and Catalysis

C

N

C

C N C

C

N

17

C C N

C

C 0.15 nm

N

C

Figure 1.17

C

N N

C C

N C C

3.6 residues

3.6 residues

(a)

C

N N

C

C

C

N

C

(b)

Describing the geometry of α-helix. The helix structure is defined by: the pitch (the distance along the

axis between successive turns) and the rise per residue. The number of residues per helical turn is 3.6. In the right handed α-helix, a complete turn of the helix contains 3.6 amino acid residues, and the distance it rises per turn (its pitch) is 0.54 nm. The R groups of each amino acid residues in an α-helix face outward (not shown in the figure). In the α-helix, the hydrogen bonds are within a single helix and are almost parallel to the helix axis.

Amino acids have different propensities for forming α-helices. Amino acid residues such as alanine, glutamine, glutamate, leucine, methionine, arginine show the higher tendency to form α-helices. Proline tends to disrupt α-helices because it lacks an –NH group and because its ring structure restricts its φ value to near –60 degrees.

β-pleated sheets β-pleated sheets form when two or more polypeptide chain segments line up side by side. Each individual segment is referred to as a β-strand. Rather than being coiled, each β-strand is fully extended. The distance between adjacent amino acids along a β-strand is approximately 3.5 Å, in contrast with a distance of 1.5 Å along an α-helix. β-pleated sheets are stabilized by interchain hydrogen bonds that form between the polypeptide backbone N—H and carbonyl groups of adjacent strands. Adjacent strand can be either parallel or antiparallel. In parallel β-pleated sheet structures, the polypeptide chains are arranged in the same direction. However in antiparallel β-pleated sheet chains run in opposite directions. Antiparallel β-sheets are more stable than parallel β-sheets because fully collinear hydrogen bonds form.

18

Biomolecules and Catalysis

O H

H

H

H

C

C

H

O

N

O

H

Ca C O

H

N

O

H

N Ca

O

O

H

C O

H

C

C-terminus

C

N

N-terminus

O

H

Ca

H

C N

N-terminus

Ca

N Ca

O Ca

N C

C

C

H

C N

Ca

N Ca

Ca

C Ca

H

C

C

N

Ca

O

Figure 1.18

O

O

Ca

C-terminus

H

H

O

C-terminus

N

N

Ca

Ca

N Ca

C

C

N

N

N-terminus

N Ca

H

N

H

C

N

C

Ca

O

C-terminus

C

O

Ca

O

H

O

O

Ca

N Ca

C

N

O

H

N-terminus

In an antiparallel β-pleated sheet, adjacent strands run in opposite directions. Hydrogen bonds between

NH and CO groups connect each amino acid to a single amino acid on an adjacent strand. In parallel β-pleated sheet, adjacent strands run in the same direction. Hydrogen bonds connect each amino acid on one strand with two different amino acids on the adjacent strand. For each amino acid, the NH group is hydrogen bonded to the CO group of one amino acid on the adjacent strand, whereas the CO group is hydrogen bonded to the NH group on the amino acid two residues farther along the chain.

Turns Most proteins have compact, globular shapes, requiring reversals in the direction of their polypeptide chains. Many of these reversals are accomplished by a common structural element called the turn. Turns, composed of three to five residues, are classified as a third type of secondary structure. These short, U-shaped secondary structures are stabilized by a hydrogen bond between their end residues. Glycine and proline are commonly present in turns. The lack of a large side chain in glycine and the presence of a built-in bend in proline allow the polypeptide backbone to fold into a tight U shape. Turns allow large proteins to fold into highly compact structures. In contrast, loops are longer than turns and do not have regular secondary structure. Turns are classified according to the separation between the two end residues participating in hydrogen bonding: α-turn, β-turn and γ-turn. In an α-turn, the donor and acceptor residues are separated by four peptide bonds (involves five amino acid residues). H-bond forms between the carbonyl oxygen of residue (n) and the hydrogen of the amide group of residue (n+4). β-turn (the most common form) is characterized by hydrogen bond(s) in which the donor and acceptor residues are separated by three peptide bonds (n and n+3). Similarly, a γ-turn is characterized by hydrogen bond(s) in which the donor and acceptor residues are separated by two peptide bonds (n and n+2). Turns are located primarily on the protein surface and thus participate in interactions between proteins and other molecules. Supersecondary structures Many globular proteins contain combinations of α-helix and β-pleated secondary structures. Specific geometric arrangements of α-helices and β-strands connected through loops are called supersecondary structures (also called motifs). These structures can be αα (two α-helices linked by a loop), ββ (two β-strands linked by a loop), βαβ (two parallel β-strands are connected by an α-helix) or more complexes structures, like the Greek key motif or the beta-barrel.

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Biomolecules and Catalysis H

H

O

N

C

C

25

(CH2)2 H

N

H

C

O

C

CH2 CH2

H2C

CH2

CH2

N

H

C

H

C

O

+

N

CH2 (CH2)3

Figure 1.22

1.2.3

N

C

C

H

H

O

Intramolecular desmosine cross-links in elastin.

Keratins

Keratins are fibrous proteins present in eukaryotes. They form a large family, with about 30 members being distinguished. Keratins have been classified as either α-keratins or β-keratins. Proteins

α-keratin

β-keratin

Characteristics

Tough, insoluble

Soft, flexible

Conformation

Helical

Extended chain

Basic unit

Protofibril

Antiparallel β-pleated sheet

α-keratins are intermediate filament proteins present only in many metazoans, including vertebrates. In vertebrates, α-keratins constitute almost the entire dry weight of hair, wool, feathers, nails, claws, scales, horns, hooves, and much of the outer layer of skin. The α-keratin polypeptide chain which forms polymerized α-keratin structure, is a right-handed α-helix and rich in hydrophobic amino acid residues Ala, Val, Leu, Ile, Met and Phe. Every α-keratin polypeptide chain dimerizes to form heterodimer. The heterodimer is made up of type I (acidic) and the type II (neutral/basic) α-keratin polypeptide chains. The two chains in heterodimer have a parallel arrangement. Two heterodimers join in an antiparallel manner to form the fundamental tetrameric subunit (a protofilament). Two protofilaments constitute a protofibril. Four protofibrils constitute a microfibril, which associates with other microfibrils to form a macrofibril.

1.2.4

Myoglobin

Myoglobin (Mb), a globular protein, contains a single polypeptide chain of 153 amino acid residues (molecular weight 17,800), and a single heme group. The inside of myoglobin consists almost exclusively of nonpolar residues, whereas the outside contains both polar and nonpolar residues. About 75% of the polypeptide chain is α-helical. There are eight helical segments. These eight helical segments are commonly labeled A–H, starting from the NH2terminal end. The interhelical regions are designated as AB, BC, CD,..., GH, respectively. The iron atom of the heme is directly bonded to a nitrogen atom of a histidine side chain of globin. Heme Globin of Mb binds a single heme group by forming a co-ordinate bond. The heterocyclic ring system of heme is a porphyrin derivative. The porphyrin in heme is known as protoporphyrin IX. It is made up of 4-pyrrole ring and 4-pyrroles are linked by methine (=CH–) bridges to form a tetrapyrrole ring. The Fe atom is present either in Fe2+ or Fe3+ oxidation state in the center of the protoporphyrin IX ring.

26

Biomolecules and Catalysis

Only myoglobin in Fe2+ state can bind O2. The iron atom has six co-ordination bond, 4 in the plane of flat porphyrin ring and two perpendicular to it. The iron atom of the heme is directly bonded to one of the histidine called proximal histidine (His93 or His F8) of globin protein. It is 93rd amino acid residue from the amino-terminal end of the

myoglobin polypeptide chain (i.e. His93) or the 8th residue in helix F (i.e. His F8). The O2-binding site is present on the other side of the heme plane, at the sixth coordination position. A second histidine residue, termed as distal histidine, His64 or His E7, (not bonded to the heme) makes H-bond with oxygen molecules. O2 binds directly to

the iron atom of the heme only. CH2 M

V

Distal histidine

CH

CH3

N N H

M N

2+

Fe

N V

H N

H

CH3

CH3

N

P

N

N M

N

O

CH

CH2

O

N CH2

N

CH2

N Fe

P

COOH

M

N

CH2

Heme N

CH3

N

P : Propionic; V : Vinyl; M : Methyl

CH2 N

Protoporphyrin IX COOH

Proximal histidine

Heme

Figure 1.23

The pyrrole rings and methylene bridge carbons are coplanar, and the iron atom resides in almost the

same plane. The fifth and sixth coordination positions of iron atom are directed perpendicular to—and directly above and below—the plane of the heme ring. The fifth coordination position of the iron is linked to a ring nitrogen of the proximal histidine. The distal histidine lies on the side of the heme ring opposite to proximal histidine.

Binding of oxygen to Mb

Myoglobin, functions as an oxygen-storage protein, binds with one oxygen molecule. The dissociation of the myoglobin-oxygen complex (MbO2) is described by K

d ˆˆˆˆ† MbO2 ‡ˆˆˆˆ Mb + O2

Kd is the dissociation constant and used to express the affinity of a protein for a ligand. A lower value of Kd corresponds to a higher affinity of ligand for the protein. Kd is given by: Kd =

[Mb][O2 ] [Mb][O2 ] or [MbO2 ] = [MbO2 ] Kd

The fractional saturation (Y) of myoglobin Y=

[Mb][O2 ] / Kd [MbO2 ] [O2 ] Number of binding sites occupied = = = Total number of binding sites [MbO2 ] + [Mb] [Mb][O2 ] / Kd + [Mb] [O2 ] + Kd

When all of the myoglobin molecules in solution are bound to oxygen, Y = 1.0. Myoglobin is saturated with oxygen at this point. When Y = 0.5, myoglobin is half-saturated with oxygen. Kd is equal to the [O2] at which half of the myoglobin molecules in solution are occupied or [O2]0.5. Y=

[O2 ] [O2 ] + [O2 ]0.5

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Biomolecules and Catalysis

31

Ascent to high altitude triggers a substantial rise in BPG concentration in red cells, with a consequent increase in the availability of oxygen to tissues. The high altitude-induced increase in BPG causes decrease in hemoglobin’s oxygen affinity. At sea level, the difference between arterial and venous pO2 is 70 torr and hemoglobin unloads 38% of its bound O2. However, when the arterial pO2 drops to 55 torr, as it does at an altitude of 4500 m, hemoglobin would be able to unload only 30% of its O2. High-altitude adaptation (which decreases the amount of O2 that hemoglobin can bind in the lungs but, to a greater extent, increases the amount of O2 it releases at the tissues) allows hemoglobin to deliver a near-normal 37% of its bound O2. Similarly, the affinity of fetal hemoglobin (hemoglobin F) for O2, which is greater than that for adult hemoglobin (hemoglobin A), facilitates the movement of O2 from the mother to the fetus. The cause of this greater affinity is the poor binding of BPG by the γ-polypeptide chains that replace β-chains in fetal hemoglobin. Effect of temperature: A rise in temperature shifts the curve to the right. Conversely, a fall in temperature shifts

the curve to the left, and a lower pO2 is required to bind a given amount of O2. Left shift (Higher affinity) Decreased temperature Decreased BPG concentration Increased pH

Y (fractional saturation)

1.0 Figure 1.28 Effect of pH, BPG Right shift (Lower affinity) Increased temperature Increased BPG concentration Decreased pH

0.5

concentration and temperature on the oxygen affinity of hemoglobin. Change in these factors shift the entire oxygenhemoglobin dissociation curve

0.0

either to the left (higher affinity) 0

50

100

or to the right (lower affinity).

pO2 (torr)

Carbon dioxide transport by Hb

Carbon dioxide binds to hemoglobin and forms carbaminohemoglobin. It combines with the amino groups of N-terminal amino acids of α- and β-globin chains. Hemoglobin transports only about 23% of carbon dioxide. The greatest percentage of carbon dioxide (about 70%) is transported through blood plasma as bicarbonate ions. Sickle-cell hemoglobin (HbS) HbS forms as a result of a single amino acid substitution in the β-chain of Hb. Replacement of the glutamate residue at position 6 in the β-chain by a valine residue is the only chemical difference between HbA and sickle-cell hemoglobin. This residue is present on the outer surface of the molecule. The change produces a sticky hydrophobic spot on the surface that results in abnormal quaternary association of hemoglobin. This makes the deoxyHbS less soluble than deoxyHbA. Insoluble deoxyHbS forms polymers that aggregate into tubular fibers. The formation of insoluble deoxyHbS fibers distorts the RBC into the elongated sickle shape structure which is the characteristic of the disease, sickle-cell anemia. Sickle-cell anemia and sickle cell trait are different. Sickle cell trait describes a condition in which an individual has one abnormal allele of β-globin gene (heterozygous). A person with sickle cell anemia has two copies of abnormal β-globin gene (homozygous).

1.2.6

Models for the behavior of allosteric proteins

Several models have been proposed to explain the behavior of allosteric proteins/enzymes. The concerted model proposed by Monod, Wyman and Changeux (MWC) and the Sequential model suggested by Koshland, Nemethy and Filmer (KNF) have been the most popular. Both these models explain the non-hyperbolic kinetics by assuming

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Biomolecules and Catalysis 1.3

33

Protein folding

Protein folding is the physical process by which a polypeptide folds into its characteristic and functional threedimensional conformation. The correct three-dimensional structure is essential to protein function. Failure to fold into native structure produces inactive proteins. A protein molecule folds spontaneously during or after biosynthesis. However, the process also depends on the nature of solvent, the concentration of salts, the temperature, and the presence of molecular chaperones. One of the most important experiment, which helped in understanding the process of protein folding was carried out by Christian Anfinsen and colleagues in the early 1960s. C. Anfinsen studied the refolding of protein ribonuclease A. Ribonuclease A isolated from bovine pancreas is an enzyme that has a molecular mass of 13,700 Da. It contains 124 amino acid residues and four disulfide linkages. In the presence of urea, a denaturant, and β-mercaptoethanol, a reducing agent, ribonuclease is denatured and the disulfide bonds are broken. When the protein is allowed to renature by removing the denaturant and the reductant, the protein regains its native conformation, including four correctly paired disulfide bonds. This finding provided the first evidence that the amino acid sequence of a polypeptide chain contains all the information required to fold the chain into its native three dimensional structure. However, when the reductant is removed while the denaturant is still present, the disulfide bonds are formed again in protein but most of the disulfide bonds are formed between incorrect partners. This indicates that weak interactions are required for correct positioning of disulfide bonds and assumption of the native conformation. Oxidation of the sulfhydryl group in the absence of urea and b-mercaptoethanol

Native ribonuclease

8 M urea b-Mercaptoethanol

Native ribonuclease

Figure 1.31

Denatured ribonuclease

Oxidation of the sulfhydryl group in the presence of 8 M urea

Scrambled ribonuclease

Denaturation and renaturation of ribonuclease. Depending on the conditions for renaturation, we obtain

either native ribonuclease or scrambled ribonuclease.

The amino acid sequence of a protein determines its native conformation. But, if this is true, how do proteins find the right conformation out of the simply endless number of potential three-dimensional forms that it could randomly fold into? After all, the folding of a protein is not a chemical reaction. The folding pathway of a polypeptide is very complicated, and not all the principles that guide the process have been worked out. However, there are several models to explain folding. According to one model, folding is initiated by a spontaneous collapse of the unfolded polypeptide chain into a partly organized globular state, mediated by hydrophobic interactions among nonpolar residues (hydrophobic collapse). The collapsed state is referred to as a molten globule. This state is clearly different from the native and the denatured state. The molten globule has most of the secondary structure of the native state but it is less compact and the proper packing interactions in the interior of the protein have not been formed. This event is very fast, usually completes within a few milliseconds. We therefore know almost nothing about the process that leads to the molten globule. However we know some of the properties of this state. As mentioned above the molten globule has most of the secondary structure of the native state. It is less compact

34

Biomolecules and Catalysis

than the native structure and lacks the proper packing interactions in the interior of the protein. The interior side chains remain mobile, more closely resembling a liquid than the solid-like interior of the native state.

Fast

Slow

Molten globule

Folded

Unfolded Figure 1.32

The molten globule state is an intermediate state in the folding pathway when a polypeptide chain

converts from an unfolded to a folded native state.

1.3.1

Molecular chaperones

Not all proteins fold spontaneously after or during synthesis in the cell. Folding of many proteins requires molecular chaperones. Molecular chaperones are a class of proteins which bind to incompletely folded or unfolded proteins in order to assist their folding or prevent them from aggregating. Chaperones function mainly by preventing formation of incorrect structures rather than by promoting formation of correct structures. Chaperones may also be required to assist the refolding of stress-denatured proteins, formation of oligomeric structures, protein trafficking through membranes and assistance in proteolytic degradation. Molecular chaperones were first identified in bacteria E. coli but are present in both prokaryotes and eukaryotes (ubiquitous). Several molecular chaperones are included among the heat-shock proteins (hence their designation as Hsp), because they are synthesized in increased amounts after a brief exposure of cells to an elevated temperature (for example, 42°C for cells that normally live at 37°C). Chaperones are usually classified according to their molecular weight (Hsp40, Hsp60, Hsp70, Hsp90, Hsp100 and the small Hsps). There are two major families of molecular chaperones known as the Hsp60 and Hsp70 families. The members of these two chaperone families function differently. The members of Hsp70 family (Hsp70, Hsc70, Hsp40 and GrpE) act early in the life of many proteins, binding to a string of about seven hydrophobic amino acids before the protein leaves the ribosome. The Hsp70 polypeptide chain is divided into two functional regions, one that binds and hydrolyses ATP and a second that binds hydrophobic segments of unfolded polypeptide chains. The polypeptide binding domain is an antiparallel C-terminal region. Hsp70 is induced by stress (e.g. heat shock) whereas Hsc70 is constitutively expressed in cells. Cytosolic Hsp70s prevent misfolding and maintain the polypeptide chain in unfolded condition. Cytosolic Hsp70s are also necessary for normal translocation of protein from cytosol into either ER or mitochondria. Hsp70 (DnaK in E. coli) works in tandem with Hsp40 (DnaJ in E. coli). The ATP-dependent reaction cycle of Hsp70 is regulated by the Hsp40. Hydrolysis of ATP to ADP is strongly accelerated by Hsp40. The Hsp60 family of molecular chaperones (also called chaperonins) forms a large barrel-shaped structure that acts later in a protein’s life, after it has been fully synthesized. Chaperonins bind unfolded, partly folded and incorrectly folded protein molecules but not protein in their native state. This type of chaperone forms an isolation chamber into which misfolded proteins are fed, preventing their aggregation and providing them a favorable environment to refold. The typical structure is a ring of many subunits, forming a cylinder. Hsp60 itself in eukaryotes (GroEL in E. coli) forms a structure consisting of 14 subunits that are arranged in two heptameric rings stacked on top of each other in an inverted orientation. This structure associates with a ring shaped heptamer formed of subunits of Hsp10 (GroES in E. coli), also described as co-chaperonin.

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47

Biomolecules and Catalysis 1.5

Nucleic acids

Nucleic acid was first discovered by Friedrich Miescher from the nuclei of the pus cells (Leukocytes) from discarded surgical bandages and called it nuclein. Nuclein was later shown to be a mixture of a basic protein and a phosphoruscontaining organic acid, now called nucleic acid. There are two types of nucleic acids (polynucleotides): ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).

1.5.1

Nucleotides

The monomeric units of nucleic acids are called nucleotides. Nucleic acids therefore are also called polynucleotides. Nucleotides are phosphate esters of nucleosides and made up of three components: 1.

A base that has a nitrogen atom (nitrogenous base)

2.

A five carbon sugar

3.

An ion of phosphoric acid

Nitrogenous bases Nitrogenous bases are heterocyclic, planar and relatively water insoluble aromatic molecules. There are two general types of nitrogenous bases in both DNA and RNA, pyrimidines and purines. H C6

H C4

7 5

1N

C

N CH

2

HC

C 4

N 3

5

3N

8

CH

2

HC

N9 H

CH 6

N 1

Purine

Pyrimidine

Purines

Two different nitrogenous bases with a purine ring (composed of carbon and nitrogen) are found in DNA. The two common purine bases found in DNA and RNA are adenine (6-aminopurine) and guanine (6-oxy-2-aminopurine). Adenine has an amino group (–NH2) on the C6 position of the ring (carbon at position 6 of the ring). Guanine has an amino group at the C2 position and a carbonyl group at the C6 position. Pyrimidines

The two major pyrimidine bases found in DNA are thymine (5-methyl-2,4-dioxypyrimidine) and cytosine (2-oxy-4aminopyrimidine) and in RNA they are uracil (2,4-dioxypyrimidine) and cytosine. Thymine contains a methyl group at the C5 position with carbonyl groups at the C4 and C2 positions. Cytosine contains a hydrogen atom at the C5 position and an amino group at C4. Uracil is similar to thymine but lacks the methyl group at the C5 position. Uracil is not usually found in DNA. It is a component of RNA.

C N

NH2

O

NH2 C

C

N

HN

C

HC

C N Adenine

N H

C

N

CH

O C CH

N

O

HN

C CH

HN

C

CH3

CH C H2N

C N Guanine

N H

C O

C

CH

N H Cytosine

O

CH N H Uracil

C O

CH N H

Thymine

Sugars

Naturally occurring nucleic acids have two types of pentose sugars: ribose and deoxyribose sugar. Ribose sugar is found in RNA. It is a five carbon monosaccharide with a hydroxyl group (–OH) on each carbon.

48

Biomolecules and Catalysis

Deoxyribose sugar is found in DNA. It is a five carbon monosaccharide, lacking one oxygen atom at 2’ position. The hydroxyl group (–OH) at 2’ position of ribose sugar is replaced by a hydrogen (–H). 5’

HOCH2

OH

O

1’

4’

H

2’

3’

HO

H

OH

b-D-Ribose

5’

HOCH2

OH

O

1’

4’

H

2’

3’

HO

H

H

b-D-2-Deoxyribose

The carbon atoms of the ribose and deoxyribose present in nucleoside or nucleotides are designated with a prime (’) mark to distinguish them from the backbone numbering in the nitrogenous bases. Unprimed numbers refer to the atoms of the nitrogenous base. Sugar pucker Pentose sugar is non-planar. This non-planarity is termed puckering. Pentose ring can be puckered in two basic conformations: envelope and twisted. In the envelope form, the four carbons of the pentose sugar are nearly coplanar and the fifth is away from the plane. In twisted form three atoms are coplanar and the other two lie away on opposite sides of this plane. Twisting the C2’ and C3’ carbons relative to the other atoms results in twisted forms of the sugar ring. Sugar pucker can be endo or exo. C2’ or C3’ endo pucker means that C2’ or C3’ are on the same side as the base and C4’-C5’ bond. Exo-pucker describes a shift in the opposite direction. Purines show a preference for the C2’endo pucker conformational type whereas pyrimidines favour C3’-endo. In RNA we find predominantly the C3’-endo conformation. 5’ C

N

5’ C

1’

O

4’

N

3’

3’

O

4’

2’

1’ 2’

Envelope form, C3’ endo 5’ C

Twisted form, C3’ endo and C2’ exo 5’ C

N

N

2’ 4’

3’

O

2’ 1’

O

4’

1’

3’ Envelope form, C2’ endo

Twisted form, C2’ endo and C3’ exo Figure 1.42

Sugar puckers.

Nucleoside Sugar and nitrogenous base join to form nucleoside. The bond between the sugar and the base is called the N-glycosidic bond. The nitrogenous base lies above the plane of the sugar when the structure is written in the

standard orientation; that is, the configuration of the N-glycosidic linkage is β.

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Biomolecules and Catalysis 1.6

53

Structure of dsDNA

Watson and Crick first described the structure of the DNA double helix in 1953 using X-ray diffraction data of DNA fibers obtained by R. Franklin and M. Wilkins. Watson, Crick, and Wilkins were awarded the 1962 Nobel Prize for Medicine for discovering the molecular structure of DNA. R. Franklin died of cancer before the Nobel prize was awarded. Nobel prizes are not awarded posthumously. The Watson-Crick double helix model describes the features of the B form of DNA. However, there are many other forms or conformations of DNA (such as A-, C-, Z-froms) which are very distinct from the B form. The form that DNA would adopt depends on several factors: the hydration level, DNA sequence, chemical modifications of the bases, the type and concentration of metal ions in solution.

1.6.1

B-DNA

B-form of DNA has following major features: •

Two long polynucleotide strands coiled around a central axis.



Strands are wrapped plectonemically in a right handed helix.



Strands are antiparallel i.e. one strand is oriented in the 5’→3’ direction and the other in the 3’→5’ direction.



Strands interact by hydrogen bonds between complementary base pairs.



G forms three hydrogen bonds with C.



A forms two hydrogen bonds with T. Major groove

Major groove

H HN–H 5

1

N Sugar

CH3

N 7

4 6

C

6

O

3

N

H–N

1

5

2

4

N

O

H– NH

N

1

N

3

N 7 6

3

N–H

N

Sugar

8

5

9

A

1

2 2

Sugar

Minor groove

Figure 1.47

H–N

4

T

6

9

G

2

5

8

O

4

N

O

N

3

Sugar

Minor groove

Standard base pairing between guanine and cytosine and between adenine and thymine via hydrogen

bonds.



Angle of interaction between base pairs result in major and minor grooves. The angle between the C1’ atoms is larger on one side than the other, generating two dissimilar grooves in the B-DNA. The side containing N7 of purines is termed the major groove, while the other side, containing N3 of purines is the minor groove.



Helix diameter is 20Å.



Helix rise per base pair is 3.32Å.



Helix pitch (distance along the axis per 360 degree turn) is 33.2Å.



10.4 base pairs per helical turn.



Base pairs are in the inside of the molecule stacked close to each other.

54

Biomolecules and Catalysis Diameter ~20 Å

Minor groove

Major groove

Helix pitch 33.2 Å

3.32 Å Axial rise

Figure 1.48

The Watson-Crick double helix is composed of about 10.4 bps per helical turn. Since 360° constitutes

one helical turn, there would be a 34.3° twist angle or rotation per residue between adjacent base pairs.

The position of the base pairs relative to the helix axis are described by another three parameters: Base pair tilts: It is a shift of the base pairs short axis relative to the vertical helix axis. The tilt angle is

measured by considering the angle made by a line drawn through the two hydrogen bonded bases relative to a line drawn perpendicular to the helix axis. The tilt angle opens in the direction of the phosphate backbone. Base pair roll: It is a shift of the base pairs long axis relative to the vertical helix axis. Propeller twist: It is the twist of the bases in a base pair against each other. A base pair is rarely a perfect

flat plane with each base in the same plane. Rather, each base has a slightly different roll angle with respect to the other base. This makes the two bases look like an airplane propeller.

Tilt

1.6.2

Roll

Propeller Twist

Z-DNA

Z-DNA is a left-handed double helical structure with two anti-parallel strands that are held together by WatsonCrick base pairing. The transition from B- to Z-DNA conformation occurs most readily in DNA segments containing alternating purines and pyrimidines, especially alternations of C and G on one strand (and also in DNA segments containing alternations of T G on one strand and C A on the other). The existence of Z-DNA was first suggested by optical studies demonstrating that a polymer of alternating C and G in one strand in a 4 M NaCl solution. The

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Biomolecules and Catalysis 1.7.2

65

RNA World hypothesis

The concept of an RNA World is a way of answering the basic problem of what was the self-replicating molecule present at the beginning of life. This hypothesis proposes that RNA was actually the first life-form on earth, later developing a cell membrane around it and becoming the first prokaryotic cell (the phrase RNA World was first used by Walter Gilbert in 1986). This hypothesis is supported by the RNA’s ability to store, transmit, and duplicate genetic information, just like DNA does and to catalyze chemical reactions, just like protein does. Because RNA can perform the tasks of both genetic materials and enzymes, RNA is believed to have once been capable of independent life.

1.7.3

RNA as genetic material

Some viruses contain an RNA as genetic material. One of the first experiments that established RNA as the genetic material in RNA viruses was the reconstitution experiment of H.Fraenkel-Conrat and B.Singer. They took two different strains of Tobacco Mosaic Virus (TMV), separated the RNAs from their protein coats, and reconstituted hybrid viruses by mixing the proteins of one strain with the RNA of the second strain, and vice versa. When the hybrid virus was spread on tobacco leaves, the lesions that developed corresponded to the TMV from which the RNA had been obtained. Thus, it was concluded that RNA serves as the genetic material in TMV.

TMV type A Infection of tobacco leaf

RNA from TMV type A and Protein (capsid) from TMV type B

TMV type A

TMV type B

Figure 1.59

In vivo reconstitution of a hybrid TMV virus. There are two strains of virus (TMV type A and type B)

which were separated into protein and RNA. The protein of one strain (type B) was allowed to recombine with the RNA of the other (type A). The in vivo progeny of this hybrid had the protein originally associated with its RNA. This proves that the genetic material of TMV is RNA, not protein.

Problem What is the approximate molecular weight of duplex DNA required to code for glyceraldehyde phosphate dehydrogenase (MW 40,000)? Solution The average molecular weight of an amino acid residue in a protein is 110. Thus, a protein whose molecular weight is 40,000 contains 40,000/110 = ~364 amino acids and requires a minimum DNA duplex of 3 × 364 = ~1090, nucleotide pairs. Since each nucleotide pair has an average molecular weight of about 650, the molecular weight of this gene would be about 1090 × 650 = 708,500. On the average, the molecular weight of coding DNA is about 18 times that of the corresponding protein. Problem The molecular weight of bacteriophage T4 dsDNA is 1.3 × 108. 1.

How many amino acids can be coded for by T4 DNA?

2.

How many different proteins of MW 55000 could be coded for by T4 DNA?

66

Biomolecules and Catalysis

Solution 1.

The genetic code is a triplet code. It will take a sequence of three nucleotides on the coding strand of DNA to specify one amino acid. The DNA of T4 contains:

1.3 ´ 108 = 2 ´ 105 nucleotide pairs = 2 ´ 105 nucleotides in the coding strand. 650

2 ´ 105 = ~ 6.7 ´ 104 codons. 3 2.

The average MW of an amino acid residue is 110. A protein of MW 55000 contains:

55000 = 500 amino acids. 110

6.7 ´ 104 codons can yield:

6.7 ´ 104 = 134. 500

Nucleic acid conversion factors Average MW of a DNA base pair = 650 Da 1 A260 unit = ~50 microgram/ml of double strand DNA 1 A260 unit = ~40 microgram/ml of single strand RNA 1 A260 unit = ~33 microgram/ml of single strand DNA 1000 bp DNA open reading frame = 333 amino acids = 37,000 Da protein To calculate the concentration of plasmid DNA in solution using absorbance at 260 nm: (Observed A260) × (dilution factor) × (0.050) = DNA concentration in μg/μl

1.8

Carbohydrates

Carbohydrates are polyhydroxy aldehydes or polyhydroxy ketones, or compounds that can be hydrolyzed to them. In the majority of carbohydrates, H and O are present in the same ratio as in water, hence also called as hydrates of carbon. Carbohydrates are the most abundant biomolecules on Earth. Carbohydrates are classified into following classes depending upon whether these undergo hydrolysis and if so on the number of products form: Monosaccharides are simple carbohydrates that consist of a single polyhydroxy aldehyde or ketone unit. Oligosaccharides are polymers made up of two to ten monosaccharide units joined together by glycosidic linkages. Oligosaccharides can be classified as di-, tri-, tetra- depending upon the number of monosaccharides present. Amongst these the most abundant are the disaccharides, with two monosaccharide units. Polysaccharides are polymers with hundreds or thousands of monosaccharide units. Polysaccharides are not sweet in taste hence they are also called non-sugars.

1.8.1

Monosaccharide

Monosaccharides consist of a single polyhydroxy aldehyde or ketone unit. Monosaccharides are the simple sugars and they have a general formula CnH2nOn. Monosaccharides are colorless, crystalline solids that are freely soluble in water but insoluble in nonpolar solvents. The most abundant monosaccharide in nature is the D-glucose. Monosaccharides can be further sub classified on the basis of: Number of the carbon atoms

Monosaccharides can be named by a system that is based on the number of carbons with the suffix-ose added. Monosaccharides with four, five, six and seven carbon atoms are called tetroses, pentoses, hexoses and heptoses, respectively.

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76

Biomolecules and Catalysis O-linked glycosidic bond

CH2OH

C

O

OH O

N-linked glycosidic bond

O

CH2

1

CH2OH

O

CH

O

Ser

OH

NH H

O

Monosaccharide

NH

C

C CH2

1

O

CH

Asn

NH H

Monosaccharide Core protein

Figure 1.67

O

Core protein

Carbohydrates are covalently attached to many different proteins to form glycoproteins. Carbohydrates

are attached either to the amide nitrogen atom in the side chain of asparagine (termed an N-linkage) or to the oxygen atom in the side chain of serine or threonine (termed an O-linkage).

1.8.8

Reducing and non-reducing sugar

Sugars capable of reducing ferric or cupric ion are called reducing sugar. A reducing sugar is any sugar that either has an aldehyde group or is capable of forming one in solution through isomerization. This functional group allows the sugar to act as a reducing agent. All monosaccharides whether aldoses and ketoses, in their hemiacetal and hemiketal form are reducing sugars. All disaccharides formed from head to tail condensation are also reducing sugar i.e. disaccharides except sucrose, trehalose are reducing sugars. All reducing sugars undergo mutarotation in aqueous solution. Disaccharides like sucrose, trehalose not capable of reducing ferric or cupric ion are called non-reducing sugar. In sucrose and trehalose, anomeric carbons of both monosaccharides participate in glycosidic bond formation. So, they do not contain free anomeric carbon atoms. Sucrose and trehalose are therefore non-reducing sugar, and have no reducing end. So it cannot be oxidized by cupric or ferric ions. In describing disaccharides or polysaccharides, the end of a chain that has a free anomeric carbon (i.e. is not involved in a glycosidic bond) is called the reducing end of the chain.

1.9

Lipids

Biological lipids are a chemically diverse group of organic compounds which are insoluble or only poorly soluble in water. They are readily soluble in nonpolar solvents such as ether, chloroform, or benzene. The hydrophobic nature of lipids is due to the predominance of hydrocarbon chains (—CH2—CH2—CH2—) in their structures. Unlike the proteins, nucleic acids, and polysaccharides, lipids are not polymers. Functions Biological lipids have diverse functions. The four general functions of biological lipids have been identified. •

They serve as a storage form of metabolic fuel.



They serve as a transport form of metabolic fuel.



They provide the structural components of membranes.



They have protective functions in bacteria, plants, insects, and vertebrates, serving as a part of the outer coating between the body of the organism and the environment.

Apart from the general functions biological lipids serve as pigments (carotene), hormones (vitamin D derivatives, sex hormones), signaling molecules (eicosanoids, phosphatidylinositol derivatives), cofactors (vitamin K), detergents (bile salt) and many other specialized functions.

Biomolecules and Catalysis 1.9.1

77

Fatty acids

Fatty acids are the simplest form of lipids and serve as constituents in a large number of complex forms of lipids. Fatty acids are long-chain hydrocarbons (4 to 36 carbons long) with one carboxyl group. Fatty acids in biological systems usually contain an even number of carbon atoms. The 16- and 18-carbon fatty acids are most common. The alkyl chain may be saturated or unsaturated. Unsaturated fatty acids may contain one or more double bonds. Fatty acids are amphipathic by nature; that is, they have both nonpolar and polar ends. O b

w

C

H3C

OH

a Hydrocarbon chain Fatty acyl chain

Figure 1.68

Structure of fatty acid.

By an older system, in a fatty acid second carbon is referred to as the α-carbon, third carbon as the β-carbon and the end methyl carbon as the ω-carbon. Table 1.16 Predominant naturally occurring fatty acids

Common name

Systematic name

Carbon atoms : Double bonds

Lauric acid

Dodecanoic acid

12 : 0

Myristic acid

Tetradecanoic acid

14 : 0

Palmitic acid

Hexadecanoic acid

16 : 0

Stearic acid

Octadecanoic acid

18 : 0

Arachidic acid

Eicosanoic acid

20 : 0

Palmitoleic acid

cis-Δ9-Hexadecenoic acid

16 : 1

Oleic acid

cis-Δ9-Octadecenoic acid

18 : 1

Linoleic acid

all cis-Δ9, 12-Octadecadienoic acid

18 : 2

Saturated fatty acid

Unsaturated fatty acid

9, 12, 15

Linolenic acid

all cis-Δ

Arachidonic acid

all cis-Δ5, 8, 11,14-Eicosatetraenoic acid

-Octadecatrienoic acid

18 : 3 20 : 4

Saturated and unsaturated fatty acids Saturated fatty acids have no double bonds in the chain. Their general formula is CH3—(CH2)n—COOH where n specifies the number of methylene groups between the methyl and carboxyl carbons. Examples of predominant saturated fatty acids are lauric, myristic, palmitic and others. Unsaturated fatty acids have one or more double bonds, and called monounsaturated or polyunsaturated respectively. The double bonds in naturally occurring fatty acids are generally in a cis as opposed to a trans configuration. The double bonds of polyunsaturated fatty acids are almost never conjugated (alternating single and double bonds).

CH2

H

H

C

C

H CH2

CH2

C

C H

Cis

Trans

CH2

78

Biomolecules and Catalysis

The systematic name includes the number of carbons, the number of double bonds, and the positions of the double bonds. For example, stearic acid (a saturated fatty acid) has 18 carbons and has the systematic name octadecanoic acid (18:0). The notation 18:0 denotes an 18 carbons fatty acid with no double bonds. Similarly, oleic acid is an 18 carbons fatty acid with one double bond and has the systematic name octadecenoic acid (18:1). An 18 carbons fatty acid with two double bonds is octadecadienoic acid (18:2). The notation 18:1 denotes an 18 carbons fatty acid with one double bond, whereas 18:2 signifies that there are two double bonds. Two systems are used for designating the position of double bonds in an unsaturated fatty acid. In carboxylreference system, fatty acid carbon atoms are numbered starting from the carboxyl terminus. The positions of the

double bonds are described by counting from the carboxyl carbon. The position of a double bond is represented by the symbol Δ followed by a superscript number. For example, cis-Δ9 means that there is a cis double bond between carbon atoms 9 and 10; trans-Δ2 means that there is a trans double bond between carbon atoms 2 and 3. In this nomenclature the carboxyl carbon is designated carbon 1. For example, palmitoleic acid has 16 carbons and has a double bond between carbons 9 and 10. It is designated as 16:1:Δ9. In omega-reference system, the position of the double bond are indicated relative to the omega carbon (i.e. number 1 is assigned to the omega carbon). For example, ω6 indicates a double bond on the sixth carbon counting from the ω-carbon. Essential fatty acids

Essential fatty acids are those fatty acids which are not synthesized by animals and must be obtained from diet. Linoleate and linolenate are the two essential fatty acids for humans and other animals. Humans lack the enzymes to introduce double bonds at carbon atoms beyond C-9 in the fatty acid chain. Hence, humans cannot synthesize linoleate and linolenate. Fatty acids that can be endogenously synthesized are termed as nonessential fatty acids. They are nonessential in the sense that they do not have to be obligatorily obtained from diet. Melting point of fatty acids

The melting point of fatty acids depends on chain length, presence or absence of double bond and number of double bonds (i.e. degree of unsaturation). The longer the chain length, the higher the melting point, and the greater the number of double bonds, the lower the melting point. The presence of double bonds makes unsaturated chain more rigid. As a result, unsaturated chains cannot pack themselves in crystals efficiently and densely as saturated chain, so, they have a lower melting point as compared to saturated fatty acids. Similarly, the unsaturated fatty acids with cis configuration have lower melting points than the unsaturated fatty acids with trans configuration. Problem Why unsaturated fatty acids have low melting points? Solution The presence of double bonds makes unsaturated chain more rigid. As a result, unsaturated chains cannot pack themselves in crystals efficiently and densely as saturated chain, so, they have lower melting point as compared to saturated fatty acids.

1.9.2

Triacylglycerol and Wax

Triacylglycerols (also called triglycerides) are triesters of fatty acids and glycerol. They are composed of three fatty acids and a glycerol molecule. Triacylglycerols are of two types – simple and mixed type. Those containing a single kind of fatty acids are called simple triacylglycerols and with two or more different kinds of fatty acids are called mixed triacylglycerols. The general formula of triacylglycerol is given below:

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84

Biomolecules and Catalysis H

O COOH H CH3

Figure 1.76

Structure of leukotriene A.

Table 1.18 Biological effects of eicosanoids

Type

Major functions

Prostaglandins

Mediation of inflammatory response Regulation of nerve transmission Inhibition of gastric secretion Sensitization to pain Stimulation of smooth muscle contraction

Thromboxanes

Platelet aggregation Aorta constriction

Prostacyclins

Thromboxane antagonists

Leukotrienes

Bronchoconstriction Leukotaxis

1.9.7

Plasma lipoproteins

Triacylglycerols, phospholipids, cholesterol and cholesterol esters are transported in human plasma in association with proteins as lipoproteins. Blood plasma contains a number of soluble lipoproteins, which are classified, according to their densities, into four major types. These lipid-protein complexes function as a lipid transport system because isolated lipids are insoluble in blood. There are four basic types of lipoproteins in human blood: chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). A lipoprotein contains a core of neutral lipids, which includes triacylglyerols and cholesterol esters. This core is coated with a monolayer of phospholipids in which proteins (called apolipoprotein) and cholesterol are embedded. Table 1.19 Some properties of major classes of human plasma lipoproteins

Lipoprotein Chylomicrons

Density (g/mL)

Protein

Phospho-

Free

Cholesterol

Triacyl-

lipids

cholesterol

esters

glycerols

Apolipoprotein