Using NMR as a Probe of Protein Structure and Function

3 Using NMR as a Probe of Protein Structure and Function

(D

n

J. P. G.Malthouse Department of Biochemistry, University College Dublin, Dublin 4, Ireland

Introduction Multi-dimensional N M R is widely used for determining the three-dimensional structure of small proteins. However, N M R can also be used as a non-invasive probe of specific features of protein structure and function. In this review I shall illustrate how we have used N M R to study a range of topics including : the stabilization of catalytic intermediates by the serine proteases, the polarity of the environment around the thiol groups in flavodoxins and P-lactoglobulins, the provenance of the thiol group of P-lactoglobulin, how the binding of flavin mononucleotide affects the environment of the thiol group of apoflavodoxins and how tryptophan synthase and serine hydroxymethyltransferase control the stereospecificity of a-proton exchange reactions. N M R is an extremely powerful and selective technique that allows us to observe signals from individual atoms in complex molecules such as proteins. However, many of the proteins that we wish to study contain at least 1000 carbon atoms and many more hydrogen atoms. Therefore one of the main problems in studying such proteins is how to pick out the signals due to the carbon atoms that we wish to study. One solution is to enrich the carbon atoms of interest with the carbon-13 isotope. This can increase the magnitude of their signals 100-fold and so enable us to identify specific carbon atoms. This is the approach used in most of the studies I shall discuss.

Delivered at Dublin City University, Dublin, on I0 September I998

J. PAUL G. MALTHOUSE much of the catalytic efficiency of the serine proteases could be due to their ability to stabilize tetrahedral intermediates [14]. However, in efficient catalysis such intermediates do not accumulate and so they cannot be observed by techniques such as N M R . Serine protease inhibitors, which form stable adducts that mimic tetrahedral intermediates, provide a solution to this problem. Specific substrate-derived chloromethane inhibitors of trypsin [5,6], chymotrypsin [7,8] and subtilisin [9-111 alkylate N-3 of the imidazole ring of the active site histidine residue in all these enzymes (Figure 1). With the use of 13C-NMR it has been shown that C-2 of these inhibitors has a chemical shift of approx. 200 p.p.m., which is typical of an sp2 hybridized carbonyl carbon. However, when these

Using 13C-NMRto study the stabilization of tetrahedral intermediates by the serine proteases Catalysis by the serine proteases proceeds via an acyl intermediate. Both the formation and breakdown of this acyl intermediate are thought to occur via a tetrahedral intermediate that is formed by the addition of the hydroxy group of the activesite serine residue to the substrate carbonyl about to be hydrolysed (Scheme 1). It is thought that

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Scheme I Formation of an acyl intermediate by the serine proteases

H-N'V

9

His

His

0

y

'7

0-

Alkylation of the active-site histidine residue of the serine proteases by chloromethane inhibitors

0

A

"q" \

HIS-57

SER-195 Figure 2 "C-NMR spectra of (a) native bchymotrypsinand (b) 8-chymotrypsin inhibited by benzyloxycarbonyl-GlyGly-[2-'3C]Phe-CH,CI I

I

I

1

I

intermediate serine residue adds to the inhibitor carbonyl carbon to form a tetrahedral adduct (Figure 3) that is analogous to the tetrahedral intermediate formed during catalysis by the serine proteases (Scheme 1). In chymotrypsin it was shown [14,16] that Met-192 was also alkylated (Scheme 2). It has been suggested that both chymotrypsin and trypsin might have evolved to stabilize zwitterionic tetrahedral intermediates and that this stabilization is an important factor in ensuring the catalytic efficiency of these enzymes [4,14-171. T h e positive and negative charges of this zwitterionic tetrahedral intermediate would be provided by the oxyanion and the imidazolium cation of the active-site serine and histidine residues. T o stabilize the zwitterionic intermediate [structure (iii) in Scheme 31 preferentially, the pK, (pK, in Scheme 3) of the oxyanion must be lowered [4] and the pK, of the imidazolium cation (pK, in Scheme 3) of the active-site histidine residue must be raised [15]. T o determine whether this proposal is correct the oxyanion and imidazolium pK, values must be determined in tetrahedral adducts formed with the serine proteases. One major limitation of X-ray crystallographic studies of transition-state analogues bound to the serine proteases is that hydrogen

Figure I

OH -cH2

II

Acyl

inhibitors alkylate trypsin [4,12,13], chymotrypsin [14,151 and subtilisin [161 the chemical shift of the C-2 carbon is approx. 100 p.p.m. (Figure 2). This demonstrates that this carbon atom is sp3 hybridized and that the hydroxy group of the active-site

R-

+ RNH2

0

intermediate

complex

I C-R'

His

TetrahedraI

ES

I

H-NAN o

o

1

Figure 3 Structure of the intact chloromethane inhibitor adducts formed with chymotrypsin, trypsin and subtilisin

OH -

R-

I

C -CH2

A

- N q N H

I

0 I

220

I

120 PPm

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\

HIS-57

Q

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atoms cannot be observed [18]. However, one major advantage of 13C-NMR is that the chemical shifts of 13C nuclei are dependent on the charge state of ionizable groups [19]. Therefore 13CN M R provides us with a powerful tool for determining the oxyanion and imidazolium cation pK, values. With inhibitor derivatives of trypsin, chymotrypsin and subtilisin, a large positive titration shift of approx. 4 p.p.m. (Table 1 ) was observed for both the 13C-enriched hemiketal carbon (Figure 4) and the 13C-enriched a-methylene carbon (Figure 5 ) . This raised the question of whether the titration shifts observed were due to the oxyanion or the imidazolium ion. T o answer this question two model compounds (Figure 6) were synthesized [ 151 that mimicked the tetrahedral enzyme chloromethane derivatives (Figure 3). In compound A both imidazole and oxyanion titrations should occur, whereas in compound B both imidazole nitrogens are alkylated so that only oxyanion titration can occur. From the titration shifts observed it was shown that oxyanion formation

produces large positive titration shifts at the hemiketal and a-methylene carbon, whereas deprotonation of the imidazolium cation produces a negative titration shift at the a-methylene carbon [15]. Similar results have been observed for model compounds having only one of these ionizing groups [20,21]. Therefore the large positive titration shifts are due to oxyanion formation. On denaturation of the chloromethane inhibitorcomplexes, the signal at approx. 100 p.p.m. due to the hemiketal carbon is replaced by a signal at 202-206 p.p.m. (pKa 5.2-5.6) characteristic of an sp2 hybridized carbon, showing that only the intact inhibitor derivative can stabilize the tetrahedral adduct [4,14,16]. However, on denaturation, the signal due to the a-methylene carbon also titrates with a pKa of 5.2-5.6, but the signal has a negative titration shift (Figure 5 ) , showing that this ionization is due to the alkylated imidazolium cation in the denatured species [15,17,20]. T h e failure to see a negative titration shift at the a-methylene carbon of intact chloromethane derivatives shows that the histidine pKa is greater

Scheme 2

Structures and chemical shifts of chloromethane derivatives of subtilisin and chymotrypsin Chymotrypeln .. Giy-193

Gly-193

x

B

R Ser-195

Ser-195

~

(1) pK, = 9.42

(2) pK, = 8.92 Ser-195

His-57

Ser-195

His-57

Subtilisin Asn-155

Asn-155

C

D

Ser-221

ser421

(1) 2-PheCMK (2) 2-Giy-Gly-PheCMK

703

Ser-221

HI.64

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than that of the oxyanion [15,17,20]. This result has been confirmed by 'H-NMR [22-241. I t has therefore been concluded that trypsin, chymotrypsin and subtilisin preferentially stabilize the

zwitterionic tetrahedral adduct and that they do this 10000-fold more effectively than analogous model compounds in water [15,17]. Studies on model compounds suggest that in chloromethane

Scheme 3

Ionizations

within

intact

chloromethane-inhibitor serine protease

derivatives

of

OH R-C

I

-Ch-lm

I

W

p 0'

OH R-

I I

R-C

C -CH2-lmH*

I I

-CH2-Im

OE (1)

R-C

I

I

-CH2-lmH*

OE (111) Table I

pKavalues and titration shifts determined from the a-met. .ylene ant

.hemiketal carbons

Abbreviations: Z, benzyioxycarbonyl; Tos, tosyl.

OH

I

~~

~~

Inhibitor

Enzyme

Carbon

Pk

Titration shift (P.P.rn.1 Reference

Z-Lys-CHlCI

Trypsin Trypsin 8-Chymotrypsin 8-Chymotrypsin Subtilisin Subtilisin

p- Hemiketal

7.88 7.99 8.85 8.99 6.92 7.04

4. I 3 4.55 4.56 4.40 4.72 3.96

Tos-Phe-CH,CI Z-Gly-Gly-Phe-CH,CI

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a-Methylene p- Hemiketal 8-Methylene p- Hemiketal a-Methylene

704

[41 [201 ~ 5 1 [151 [I61 [I73

Royal Irish Academy Medal Lecture

oxyanion hole [26-281 has been shown to make an important contribution to the catalytic efficiency of the serine proteases [1,2,29]. T h e oxyanion pKa is lowest in subtilisin chloromethane derivatives, showing that oxyanion stabilization is most effective in subtilisin (Table 2). It has been suggested that alkylation of the active-site histidine residue in chloromethane derivatives could force the oxyanion out of its optimal position to form hydrogen bonds in the oxyanion hole [21]. However, it has also been suggested that in subtilisin the side chain of Asp-155 could move to compensate for the oxyanion not being in its optimal position [16]. Such movements would not be possible in chymotrypsin and trypsin chloromethane derivatives because the hydrogen-bond donors are main-chain N H groups (Scheme 2). Therefore this movement of the side chain of Asp155 might explain why oxyanion stabilization is so much more effective in chloromethane derivatives of subtilisin [16]. However, replacement of Asp155 with an alanine residue only produced an increase of 1.09 pKa units in the oxyanion pKa [30], so that this hypothesis does not explain why the oxyanion pK, is 2-2.5 pKa units higher in chymotrypsin. T h e small effect of the Asp-155 + Ala mutation was expected because the low pKa of the oxyanion in the wild-type enzyme shows that there is a low charge density on the oxyanion;

inhibitor adducts an interaction between His-57 and Asp-102 is required to raise the pKa of His-57 so that pK, > pK, (Scheme 3) and the zwitterionic tetrahedral adduct predominates [ 15,251. Hydrogen-bonding of the oxyanion in the

Figure 4

p H titration of the I3C-enriched carbon of the intact benzyloxy~arbonyl-Gly-Gly-[2-'~C] Phe-CH,-subtilisin derivative 104 1

I

1

I

I

102

E pKa = 6.92 A p.p.m. = 4.72

P

98

3

5

7

9

11

PH

Figure 5

p H titrations of the "C-enriched carbon of the intact and denatured benzyloxycarbonyl-Gly-Gly-[ I -"C]Phe-CH,-subtilisin derivatives I

I

I

I

I

I

1

I

I

INTACT

p"

C-13C~lmW

R-

60

I

OE

/

59

2 57

55

pKa= 5.58

A p.p.m. 541 2

pKa = 7.04

A p.p.m. -3.96

= -1 -62

1

I

I

I

I

I

3

4

5

6

7

8

I

I

I

1

9

10

11

12

PH

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hydrogen-bonding to the oxyanion is therefore not expected to have a large effect on its pK8. From this increase in pK, it was estimated that hydrogen-bonding in the oxyanion hole decreased the oxyanion pK, by only approx. 2.8 pK,

units. However, it was argued that because water is a good hydrogen-bond donor a similar or larger pK, decrease would be expected in water. Therefore it was concluded that the decrease of 5 pK8 units (28.5 kJ/mol) in the oxyanion pK, in subtilisin derivatives must be due to their electrostatic interaction with the imidazolium cation of the active-site histidine residue and that the main role of hydrogen-bonding in the oxyanion hole is to provide localized solvation of the oxyanion equivalent to that which would occur in water [15,16,30]. It was estimated that hydrogen-bonding to the oxyanion stabilizes catalytic tetrahedral intermediates by approx. 35.7 kJ/mol [30]. Thus although hydrogen bonding in the oxyanion hole stabilizes catalytic tetrahedral intermediates by approx. 35.7 kJ/mol, it contributes only approx. 15.7 kJ/mol to decreasing the oxyanion pK, in chloromethane derivatives [30].

Figure 6

Model compounds

COMPOUND A

R-C

I -CHp

-N

A + NH

\-I

I I

0

CH3 Chemical modification of the thiol groups of proteins by "C-enriched cyanide and the use of the chemical shift of the thiocyanate carbon as a probe of the environment of the thiol groups

COMPOUND B

OH

Chemical modification of thiol groups in proteins by reporter groups can allow us to study the environment of the thiol group in the protein. However, fluorimetric or spectrophotometric reporter groups are usually large molecules that can often perturb the structure of the protein. In

I

I

CH3

Table 2

Stabilization of the oxyanion by subtilisin, chymotrypsin and trypsin Abbreviations: Z, benzyloxycarbonyl; Tos, tosyl.

Enzyme derivative or model

compound

Oxyanion pK,

Species (iv) in Scheme 3 Species (iii) in Scheme 3 Z-[2-'3C]Phe-CH2-6-chymotrypsin Tos-[2-'3C]Phe-CH2-6-chym~tryp~in Z-Gly-Gly-[2-' 3C]Phe-CH,-b-chymotrypsin Z-[2-'3C]Lys-CH,-trypsin Z-Gly-Gly-[2-'3C]Phe-CH2-subtilisin Ac-Leu-[ I -'3C]Phe-CF,/G-chymotrypsin

I I.9t 10.71. 9.4 8.9 8.9 7.9 6.9 d 4.9

* t

A pK,*

>

0 - 1.2 - 2.5 - 3.0 - 3.0 -4.0 - 5.0 -4.2%

Relative t o pK, in Scheme 2, i.e. ApK, = pK,- I I .9. Malthouse et al. [4], Finucane and Malthouse [IS]. f. Relative to the pK, of 9 . I for the appropriate hemiketal model compound of Liang and Abeles [21].

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Royal Irish Academy Medal Lecture

other at 115.5 p.p.m., showing that both Cys-54 and Cys-127 had been cyanylated. This showed that the thiocyanate group with a chemical shift of 109.6 p.p.m. was in a non-hydrogen-bonding apolar environment, whereas the thiocyanate group with a chemical shift of 115.5 p.p.m. was in a polar environment equivalent to an aqueous solution. On removal of the bound F M N these signals were replaced by signals at 109.4 and 112.2 p.p.m. T h e addition of F M N restored the original signals. Because neither thiol groub is near the F M N binding site it was conclude4 that these changes in chemical shift were due to cobformational changes caused by F M N binding. ' T h e signals due to cyanylated apoflavodoxin were unstable at 28 "C and were slowly replaced by signals with chemical shifts of 114.5 and 115.3 p.p.m. that were attributed to irreversibly denatured apoflavodoxin, which does not bind F M N . One possible explanation is that one thiol group is buried and is therefore affected only slightly by F M N binding, whereas the other is moved from a buried to an exposed position on F M N binding. In contrast, when the single thiol group of apoflavodoxin from C . pasteurianum was cyanylated, its thiol seemed to move to a less polar environment on flavin binding [34]. p-Lactoglobulin is a major whey protein in the milk of ruminants. Aschaffenburg and Drewry [40,41] found that bovine milk could contain either or both P-lactoglobulins A and B. These proteins have identical amino acid sequences except that in P-lactoglobulin A residues 64 and 118 are aspartate and valine respectively, whereas they are glycine and alanine respectively in P-lactoglobulin B [42]. P-Lactoglobulins A and B both contain five cysteine residues [43]. A disulphide bond exists

contrast, cyanylation of a thiol group is much less likely to perturb the structure of a protein significantly. Therefore cyanylation of cysteine residues to produce P-thiocyanatoalanine residues provides a cheap and easy way of introducing a small 13Cenriched reporter group into a protein [31-351. T h e thiocyanate carbon has a chemical shift of approx. 109 p.p.m. in a hydrophobic non-hydrogen-bonding solvent such as cyclohexane, whereas in a polar hydrogen-bonding solvent such as water it has a chemical shift of approx. 115 p.p.m. [35]. Therefore, as it is possible to resolve chemical shifts separated by only 0.1 p.p.m., this is a highly sensitive probe. I t has been used to study changes in the environments of thiols when F M N binds to apoflavodoxins from Megasphaera elsdenii [35] and Clostridium pasteurianum [34]. It has been used to resolve a conflict between X-ray crystallographic studies and chemical modification studies on Plactoglobulins [36]. Cyanylation of thiol groups can be achieved with a two-step process. First, the thiol reacts with 5,5'-dithiobis(2,2'-nitrobenzoic acid) to form a mixed disulphide, releasing 2-nitro-5-thiobenzoic acid (reaction 1, Scheme 4). From the amount of 2-nitro-5-thiobenzoic acid released we can calculate the amount of thiol modified. T h e addition of excess cyanide results in cyanylation (reaction 2, Scheme 4). From the amount of 2-nitro-5-thiobenzoic acid released on adding cyanide we can calculate the amount of thiol cyanylated. This procedure has been used to cyanylate the thiol groups of apoflavodoxin from M . elsdenii [35,37,38] and the thiol group of P-lactoglobulins A and B [36,39]. T h e cyanylated flavodoxin from M . elsdenii gave two signals [35], one at 109.6 p.p.m. and the

I

Scheme 4

Cyanylation of thiol groups

\

cool

Cool

'Cool.

+ 707

-4.. COO-

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Biochemical Society Transactions ( 1999) Volume 27, part 4

between Cys-66 and Cys-160 [44]. McKenzie et al. [45] reported that when P-lactoglobulin was incubated with ['4C]iodoacetamide at pH 8.5 in 8 M urea, equimolar amounts of Cys-119 and Cys121 were alkylated. This suggests that the 1 mol of thiol/mol of monomer consists of 0.5 mol of thiol/mol of monomer from both Cys-119 and Cys-121 [model (b) in Scheme 51. Therefore equimolar amounts of disulphide bonds involving cysteine residues 106-1 19 and 106-1 2 1 must also be present [model (b) Scheme 51. However, X-ray crystallographic studies [46,47] have shown only one disulphide bond [between residues 106 and 119; model (a) in Scheme 51 involving Cys-106, which conflicts [44] with the results of McKenzie et al. [45]. Cyanylation of the thiol group or thiol groups of P-lactoglobulin should resolve this conflict because only one thiocyanate signal should be detected if Papiz et al. [46] and Monaco et al. [47] are correct, whereas two thiocyanate signals are expected if McKenzie et al. [45] are correct. Cyanylation [36,39] of a mixture of P-lactoglobulins A and B (Figure 7a) resulted in two thiocyanate signals, one at 109.7 p.p.m. and the other at 114.4 p.p.m. (Figure 7b), which suggests that McKenzie et al. [45] are correct. Both signals were also observed when only P-lactoglobulin A (Figure 7c) or P-lactoglobulin B (Figure 7d) was cyanylated and so different signals were not obtained from P-lactoglobulins A and B. It was found that more of the signal at 114.4 p.p.m. was

observed when P-lactoglobulin A was cyanylated (Figure 7c). However, P-lactoglobulin A is more susceptible than P-lactoglobulin B to alkaline denaturation [48] and the chemical shift of the signal at 114.4 p.p.m. is similar to the value that we would expect to observe in a denatured sample [35]. This suggests that the signal at 114.4 p.p.m. is due to denatured cyanylated P-lactoglobulin. Denatured P-lactoglobulins can be removed by adjusting the pH to 5.2 [48,49]. Adjusting the pH to 5.2 removed the signal at 114.4 p.p.m. (Figure 7e), confirming that this signal was due to denatured cyanylated P-lactoglobulin. Therefore intact cyanylated P-lactoglobulin contains only the one thiocyanate signal at 109.7 p.p.m. [36], showing that the X-ray crystallographic results of Papiz et al. [46] and Monaco et al. [47] are correct. It was shown that when intact cyanylated B-lactoglobulin B was reversibly unfolded in 7.4 M urea (Figure 7f and Figure 7g) it had a chemical shift of 114.4 p.p.m. However, alkali-denatured /I-lactoglobulin A could not be refolded by this urea treatment (Figure 7h and Figure 7i). Therefore it was concluded that the signal at 114.4 p.p.m. was formed by irreversible alkaline denaturation during the cyanylation procedure [36]. Owing to the low sensitivity of 13Cnuclei it is important that we can predict their linewidths and spin lattice relaxation times so that we can determine which acquisition parameters will give an optimal signal-to-noise ratio. The linewidths and spin lattice relaxation times of the thiocyanate

Scheme 5

Two models to explain the thiol and disulphide bonds in P-lactoglobulins M d d (a) 0s-106

s

SH

C~S-119 CyS-121

0%-106

cys-106 I

Cys-I19

cys-I19 cya-121

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CyS-121

Royal Irish Academy Medal Lecture

carbon of cyanylated /I-lactoglobulin B at magnetic field strengths of 1.88, 6.34 and 11.74 T were determined [SO]. From these values the linewidths and spin lattice relaxation times of thiocyanate carbons at magnetic field strengths of 1.88-14.1 T in proteins with molecular masses of 10-400 kDa were calculated. It was concluded that for optimal resolution magnetic field strengths of no greater than 6 . 3 4 T should be used and that different magnetic field strengths are not expected to produce large differences in the resolution and sensitivity of thiocyanate carbons attached to proteins of molecular mass greater than 10 kDa [SO].

Deuteration of methylene and methine carbons to reduce linewidths when examining large biomolecules T h e full potential of using 13C-enriched methylene and methine carbons as atomic probes of proteins has not been realized. This is because such carbon atoms undergo efficient dipolar relaxation with the directly bonded protons, which results in broad signals that are difficult to resolve from those of the natural abundance signals. Replacing the directly bonded protons with deuterons can decrease the linewidths to as little as 1/16 if there is

Figure 7

"C-NMR spectra of cyanylated /34actoglobulins at pH 7.0 1

I

I

I

114.4 p.p.m.

f163'4

I

I

I

~ 1 0 9 . p.p.m. 7

zzved 1

I

Sample (c)

P'P'm' ~ 1 1 4 . P.P.m. 6

+7.4M

Urea

H 109.7 p.p.m. Urea

1

removed

+7.4 M

109.7 p.p.m.

P.P.m.

lo9m7

cyanylated 6-lactoglobulin A+B

Native 6-lactoglobulin A+B

I

200

I

1

1

I

100 6(p.p.m.1

709

I

I

I

I

A

0

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deuterium decoupling ; it could therefore diminish this problem [33]. T h e linewidths and spin lattice relaxation times of a deuterated and a nondeuterated methylene carbon rigidly attached to chymotrypsin have been determined at magnetic field strengths of 1.88 and 6 . 3 4 T [51]. T h e signal from the deuterated methylene carbon was decoupled by scalar relaxation of the second kind. How deuteration could be used to decrease linewidths in biomolecules of different molecular masses was discussed. We also showed that a spin

echo sequence could be used to remove signals due to protonated carbons without attenuating the signals due to the deuterated carbon [51].

Stereospecificity of the exchange of the a-protons of amino acids when catalysed by tryptophan synthase and serine hydroxymethyltransferase In this study we are attempting to quantify and understand the stereospecificity of the exchange of the a-protons of amino acids catalysed by pyri-

Figure 8

I3C-NMR time course of the serine hydroxymethyltransferase-catalysed exchange of the pro-2S proton of [2-"C]glycine and of the tryptophan synthase-catalysed exchange of the remaining pro-2R proton of [2"C,ZH]glycine

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doxal phosphate-dependent enzymes. Such studies are essential if we are to use existing enzymes or if we are to design new enzymes for the stereospecific biosynthesis of chemicals. After an initial study on the exchange of the aprotons of L-serine and L-tryptophan catalysed by yeast tryptophan synthase [52], a study of the stereospecificity of the exchange of the a-protons of amino acids catalysed by serine hydroxymethyltransferase from beef liver [53] and tryptophan synthase from Salmonella typhimurium [53-551 was undertaken. It was shown [53,56] that by using 13C-NMR the exchange of both the pro-

2R and pro-2S protons of [2-13C]glycinecatalysed by serine hydroxymethyltransferase and tryptophan synthase could be followed independently (Figure 8). With this procedure it was shown that earlier claims that serine hydroxymethyltransferase had absolute stereospecificity for the pro-2S proton of glycine were incorrect. It was also shown that tryptophan synthase preferentially catalyses the exchange of the pro-2R proton of glycine [5 3,561. Bacterial tryptophan synthase is an a& complex that catalyses the final two reactions in the biosynthesis of L-tryptophan [57-591. T h e asubunits catalyse the cleavage of indol-3-ylglycerol phosphate to indole and D-glyceraldehyde 3phosphate. T h e 8-subunits catalyse the formation of L-tryptophan from L-serine and indole. In the enzyme complex the a-active site and the 8active site are separated by 25-30 A and are connected by a tunnel that allows the indole produced at the a-active site to pass to the B-active site without diffusing through the bulk solvent [59]. Large ligands such as ~ , ~ - a - g l y c e r o l - 3 phosphate that bind to the a-subunits can greatly inhibit the reaction of large nucleophiles such as indole with the aminoacrylate intermediate at the 8-active site. T h e fact that ~,~-a-glycerol-3-phosphate obstructs the entrance to the tunnel connecting the a-active site and the 8-active site is thought to be a major factor in this inhibition [60,61]. This tends to mask allosteric effects on the 8-subunit owing to the ~,~-a-glycerol-3-phosphate’s binding to the a-subunits. However, amino acids such as glycine and L-serine do not pass through the tunnel but pass directly from the bulk solvent to the /?-active site [61]. Therefore by

Figure 9

Schiff base formed between an L-amino acid and pyridoxal phosphate

H I

-0oc- ‘c1R, I 1.

-ah +N H’ *CH I

I

CH,OPi

CHS

N

I

H+

SdrCMncrr Some possible binding modes of amino acid-pyridoxal phosphate Schiff bases to enzymes The rectangle represents the plane of the imine-cofactor x-electron system, which is perpendicular t o the plane of the paper The amino acid of the Schiff base of pyndoxal phosphate is viewed along the bond linking the a-carbon and a-nitrogen of the amino acid.

(a) L-amino acid

(b) D-amino acid

71 I

(c) D-amino acid

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Biochemical Society Transactions ( 1999) Volume 27, part 4

stereospecific syntheses of L-[1 ,2-'3C,,'5N]serine and L-[l ,2-'3C,,'5N]tryptophan [64].

studying the properties of such amino acids when they are bound at the /?-active site the allosteric effects caused by the binding of D,L-a-glyCerOl-3phosphate to the a-subunits can be isolated and characterized. By using 13C-NMR it was shown that the /Iz subunits of tryptophan synthase preferentially catalysed the exchange of the pro-2R proton of glycine but adding a-subunits decreased the stereospecificity of the exchange reaction. Also adding ~,~-a-glycerol-3-phosphatecaused a further decrease in the stereospecificity of exchange [55]. Dunathan [62] has argued that for optimal catalytic efficiency the a-carbon bond to be cleaved should be orthogonal to the plane of the iminecofactor n-electron system (Figure 9). Therefore the stereospecificity of the exchange of the a-protons of L and D amino acids or of the pro-2R or pro-2S protons of glycine depends on how the carboxylate and R groups are bound (Scheme 6) relative to the plane of the imine-cofactor nelectron system [62,63]. Similarly, for the exchange of a-protons we would expect the degree of stereospecificity to depend on the efficiency of binding of the amino acid's R group and acarboxylate group by an enzyme (Scheme 6). Therefore to determine how different side chains contribute to the stereospecificity of tryptophan synthase-catalysed exchange reactions we have examined the exchange rates of the a-protons of glycine, L- and D-alanine and L- and D-tryptophan [54]. Increasing the size of the R-group led to a progressive increase in the stereospecificity of the exchange reaction. The observed increases in stereospecificity were largely due to a decrease in the first-order exchange rate of the slowly exchanged D-protons as the size of the R-group increased. This suggests that the increase in stereospecificity was due to the larger side chains of these D-amino acids being restricted in their ability to be bound in a conformation that favours exchange of the a-proton [54]. From these studies we concluded that both the a-carboxylate group and a large side chain such as that of tryptophan can make similar large contributions to the stereospecificity of a-proton exchange reactions [54,55].

This work was supported by grants from Forbairt, Cambridge Isotope Laboratories 2nd Research Grant Program and the COSTD 7 'Molecular Recognition Chemistry' programme of the EU. I thank all my co-workers and collaboratorswho are co-authors on the cited papers. The Royal Irish Academy Medal Lecture is sponsored by the Keny Group plc. I Asboth, B. and Polgar, L. ( 1983) Biochemistry 22, I 17- I22 2 Bryan, P., Pantoliano. M. W.. Quill, S. G., Hsiao, H. Y. and Poulos, T. (I 986) Proc. Natl. Acad. Sci. U.S.A. 83, 3743-3745 3 Wells, J. A., Cunningharn, B. C., Graycar, T. P. and Estell, D. A ( I 986) Phil. Trans. R SOC. Lond. A 3 I 7 , 4 I 5 4 2 3 4 Matthouse, J. P. G., Primrose, W. U., Mackenzie. N. E. and Scott, A. I. ( I 985) Biochemistry 24, 3478-3487 5 Shaw, E. and Springhorn, S. ( I 967) Biochem. Biophys. Res. Commun. 27, 39 1-397 6 Coggins, J. R, Kray. W. and Shaw, E. ( I 974) Biochem. J. 138,579-585 7 Ong, E. B., Shaw, E. and Schoellrnann, G. ( I 965) J. Biol. Chern. 240, 694-698 8 Schoellmann, G. and Shaw, E. ( I 963) Biochemistry 2, 252-255 9 Shaw, E. and Ruscica, J. ( I 968) J. Biol. Chem. 243, 63 12-63 I 3 I0 Morihara, K. and Oka, T. ( I 970) Arch. Biochem. Biophys. 138,52653 I I I Powers, J. C., Lively, M. 0.and Tippett, J. T. ( I 977) Biochirn. Biophys. Acta 480,24626 I I 2 Malthouse, J. P. G., Mackenzie, N. E., Boyd, A. S. F. and Scott, A. I. ( 1983) J. Am. Chern. SOC. 105, 1685- I 686 13 Scott, A. I., Mackenzie. N. E., Matthouse, J. P. G., Primrose, W. U., Fagemess, P. E., Brisson, A,, Qi, L. Z.. Bode, W.. Carter, C. M. and Jang,Y. J. ( 1986) Tetrahedron 42, 3269-3276 14 Finucane, M. D., Hudson, E. A. and Matthouse, J. P. G. (1989) Biochern. J. 258.853-859 I5 Finucane, M. D. and Malthouse, J. P. G. ( I 992) Biochem. J. 286,889-900 I 6 O'Connell, T. P. and Matthouse, J. P. G. ( I 995) Biochern. J. 307, 353-359 I 7 O'Connell, T. P. and Matthouse, J. P. G. (I 996) Biochem. SOC. Trans. 24, I355 I 8 Kossiakoff, A. A. and Spencer, S. A. ( I 98 I ) Biochemistry 20, 6462-6474 I9 Jardetsky,0.and Roberts, G. C. K. ( I 98 I ) NMR in Molecular Biology, pp. 28 1-287, Academic Press, New York 20 Primrose, W. U., Scott, A. I., Mackenzie, N. E. and Malthouse, J. P. G. ( I 985) Biochem. J. 23 I,677-682 2 I Liang, T. C. and Abeles, R H. ( I 987) Biochemistry 26, 7603-7608 22 Robillard, G. and Shulman, R G. ( I 972) J. Mol. Biol. 71, 507-51 I 23 Robillard, G. and Shulrnan, R G. ( I 974) J. Mol. Biol. 86, 5 19-540 24 Tsilikounas, E., Rao, T., Gutheil, W. G. and Bachovchin, W. W. ( I 996) Biochemistry 35,2437-2444 25 Rogers, G. A. and Bruice, T. C. ( I 974) J. Am. Chem. SOC. 96,2463-248 I 26 Henderson, R ( I 970) J. Mol. Biol. 54, 34 1-354 27 Kraut, J. (I 977) Annu. Rev. Biochem. 46, 33 1-358

Biosynthesis of isotopically enriched compounds Isotopically enriched compounds have important applications in structural studies of proteins by NMR. Serine hydroxymethyltransferase and tryptophan synthase have been used to catalyse the

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