Enzyme-Substrate Interactions Structure of Human ...

Reprinted from J. Mol. Biol. (1994) 241, 226-232

Enzyme-Substrate

Interactions

Structure of Human Carbonic Anhydrase I Complexed with Bicarbonate Vinay Kumar and K. K. Kannan

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J. Mol. Biol. (1994) 241, 226-232

Enzyme-Substrate

Interactions

Structure of Human Carbonic Anhydrase I Complexed with Bicarbonate Vinay Kumar and K. K. Kannan Solid State Physics Division Bhabha Atomic Research Centre Bombay-400085, India The structure of HCAI-HC03 complex has been refined with 1O-1'6AX-ray diffraction data to an R-value of 17'7%. The structure reveals monodentate binding of the HC03 anion at an apical tetrahedral position to the zinc ion. The binding mode and interactions of HC03 in HCAI differ from that in HCAII. The activity linked H20/OH- group in the free HCAI is replaced by the hydroxyl group of the bicarbonate anion. This result rules out the rearrangement ofthe bound HC03 advocated earlier to explain the microscopic reversibility of the catalysed reaction. From the geometry of the H-bonds between Glu106-Thrl99 pair and Glu1l7-His1l9 couple, the glutamic acids are expected to be ionized and accept H-bonds from their partners. The product-inhibition by HC03 anion is explained on the basis of proton localization on His1l9 in the Glu1l7-His1l9 couple. These results are consistent with the hypothesis that Glu1l7-His1l9 tunes the ionicity of the Zn2+ and the binding strength of HC03 anion. A 1t hydrogen bond is observed between a water and phenyl ring of the Tyrl14 residue. Keywords: carbonic anhydrase structure; bicarbonate; substrate binding; product-inhibition; synchrotron radiation

1. Introduction

Bicarbonate anion, substrate for the reverse reaction, inhibits the C02 hydration reaction at high pH (Steiner et at., 1975; Pocker & Deits, 1983). Recently, structures of a Thr200His mutant of human carbonic anhydrase II (HCAII) and Cobalt substituted HCAII complexed with HC03 at basic pH have been refined at 1.9 A resolution (Xue et at., 1993; Hakansson & Wehnert, 1992). The binding affinity of HC03 to the mutant and the Co(lI) substituted HCAII differs from that of human carbonic anhydrase I (HCAI) (Behravan et at., 1990; Kogut & Rowlett, 1987). Also, the rate of interconversion of C02 and HC03 for CAI, probably dissociation of HC03, can limit the overall catalysis (Simonsson et at., 1982) in contrast to that of CAlI. We report the first high resolution structure of HCAI com piexed with HC03.

Carbonic anhydrases (CAt) (carbonate dehydratase, EC 4.2.1.1) are zinc metalloenzymes that catalyse the hydration of C02' The forward reaction, hydration of C02' is catalysed at basic pH whereas the reverse dehydration of HC03 is promoted at acidic pH. The catalysed reaction has been proposed to proceed in two steps (Lindskog et at., 1984). For the C02 hydration reaction, it is believed that deprotonation of zinc-bound water produces an OH- which carries a nucleophilic attack on the substrate C02' leading to the product HC03. During the CO2-HC03 exchange reaction the specific interactions of the substrate with CA are important in elucidating the details of the reaction pathway (Liang & Lipscomb, 1987). The co-ordination geometry of the metal ion also seems to influence the catalytic turn over as substitution of Zn2+ with C02+ lowers the activity by half and that with other transition metal ions like C03+, Cu2+, Ni2+, abolishes the catalytic activity. From theoretical calculations and spectroscopic studies the substrate has been proposed to bind in the vicinity of the zinc ion.

2. Experimental (a) Data collection Crystals of the complex were obtained by soaking native HCAl crystals in solution containing 1.0 M NH4HC03 in 2'5 M (NH4}zS04' 0.05M Tris-S04, (pH 8'7). The unit cell parameters were a=81'85 A, b=75.31 A, C=37.76 A, and the space group P212121. The crystals are isomorphous with the native HCAl (Kannan et al., 1989).

t Abbreviations used: CA, carbonic anhydrase; HCAII, human carbonic hydrase II. 226 0022-2836/94/320226-07

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Human Carbonic Anhydrase I-Bicarbonate Complex Table 1 Stereochemical parameters obtained using P ROC HE K (Laskowski et al., 1993) and data statistics in HCAI-HCO:; structure Parameter value Data statistics 1'6

Resolution (A) Unique obervations Completeness (%) Toal no. of atoms

24,976 79'1 2282 2024 8.17 17'7 0'1

No. of protein atoms Rm..g. (%) Refinement R-factor Error in co-ordinates using Luzzatti

plot (A)

Stereochemical

parameters

% residues

89'7

in the core

rJ>,f region Bad contactsl100 residues 'i, (gauche +) \, (gauche -) 1., (trans) esd of pooled I, rJ>(proline) Helix angle, phi Helix angle, psi H-bond energy (kcal/mole) Trans omega C. -N -c-cP

0'4 -64'1 :t9'9 +64'1 :t9'7 18H:t 13.1 10'3 -64'6:t 11.8 -63'0:t 12'0 - 37'6:t 9'7 - 2.0 :t 0'7 179'4 :t 6'2 33'8:t 2.8

The intensity data were collected on Sakabe's weissenberg Camera (Sakabe, 1983) using Fuji imaging plate system and synchrotron radiation at Photon Factory, .Japan. and processed with the WEISS suite of programs (Higashi, 1989). The observed data extending to 1'6 A resolution with an Rmergeof 8'17%, is complete up to 79'1 % (Table 1). (b) Refinement The structure was refined by the stereochemically restrained least squares method (PROLSQ) (Hendrickson & Konnert, 1980) and manual model editing carried out on an IRIS 4D/20 computer graphics system using FRO DO/TOM suite of programs (Jones, 1982; Cambillau & Horjales, 1987). The HCAI structure (Protein Data Bank (Bernstein et at., 1977) entry 2CAB (Kannan et at., 1984)), without solvent molecules, refined against 2 A data was used as an initial model. The refinement was initiated at 3,0 A resolution with an overall B-value for the model. The diffraction data were extended in steps to include data up to 1.6 A resolution. Bicarbonate anion and solvent molecules identified from difference Fourier maps were refined initially with unit occupancy and individual isotropic B-values. Subsequently, occupancy of bicarbonate anion and solvent molecules were refined, in addition to positional parameters and individual B values for all the atoms. Parameters for bicarbonate anion and partially overlapping solvent molecules were refined simultaneously after removing the van der Waals restraints. Only solvents with refined occupancy of more than 30%, or B-value of less than 60 A2 were retained in the model. The stereochemistry of the final model was evaluated using the PROCHECK suite of programs (Laskowski et at., 1993).Co-ordinates, and structure factor amplitudes

227

and phases have been deposited with the Protein Data Bank (IHUB), from which copies are available (Bernstein et at., 1977).

3. Results The structure of the complex has been refined against data in the resolution range of 10 to 1.6 A to a crystallographic R-factor of 17'7 % (Table I). The stereochemistry of the model is good, with an average error of about 0'1 A as expected from the Luzzati plot (Luzzati, 1952). Except for the two NH2-terminal residues and side-chains of Asp4, Asp9, LyslO, Leu19 and Lys149, most of the residues have well defined electron densities. Mean B-value for the protein atoms and solvent molecules is 16.2 A2 and 28.9 A2 respectively. The B value for the bicarbonate anion dropped from 35 A2 to 17 A2 when the occupancy was refined to 40%. There was however no significant change in the positional parameters. Consistent with the chemical sequence of HCAI (Hemett-Emmett et al., 1984), Fourier maps and H-bond criteria, residues 74 and 75 were corrected, from the PDB entry of Gin and Asp to Asp and Asn, respectively. The r.m.s. displacement between the native model (Kannan, 1990), and the present complex structure is 0.2 A and 0,6 A, respective~ for the main-chain atoms and all the protein atoms. The somewhat larger r.m.s. displacement for all the equivalent atoms is due to conformational shift in the side-chains of about 20 residues not forming the active site of CAT. No significant changes are observed in His94, His96 and His 119 residues, which form the base of the Zn2+ tetrahedron, and residues constituting the active site except for marginal shifts in the side-chains of Leu 198 and His200. The H-bond network extending from zinc bound OH- group to His200 through Thrl99, GlulO6, Tyr7, His64, His67 and a number of solvent molecules, as observed in the native structure (Kannan et al., 1984), is conserved in the complex (Table 2) except that the OH - group of HCO:; replaces the OH- group present in the native HCAT. However, H-bonds of functional importance between OE2 and OG I atoms of invariant Glu 106 and Thrl99 residues (Kannan et al., 1977; :Vlerz, 1990), and OEI and NE2 atoms of Glu117 and His119 (Krauss & Garmer, 1991) are marginally stretched to 2.8 A as compared to 2.6 A in the native HCAI (Table 2), and also in HCAIT, buffalo CAIT, bovine CAllI and a number of complexes of HCAT

and HCAII (Hakansson et al., 1992; Vinay Kumar et al., 1989, 1994; Eriksson & Liljas, 1993). The resulting residual valence on the OEI atom of Glu1l7 is satisfied by a strong H-bond from KDl atom of Hisl07 (d=2'8A). Based on an analysis of high-precision neutron structures of amino acids and small peptides in the Cambridge Data Base, it has been observed that the hydroxyl of a carboxylic group cannot accept a H-bond and a distance of more than 2'7 A signifies an ionized carboxylic group (Ramanadham et al., 1993). Earlier a shut-

228

Important Tyr70H Tyr7 OH His64 ND I His 64 NE2 8er65 OG 8er65 OG His67 NE2 His67 NDl Gln92 OEI Gln92 OEI Gln92 OEI Glul06 N GlulO6 OE2 GlulO60EI Glul06 OEI His 107 NDl Glull7 OEI Thrl99 N Thrl990GI Thr 199 om His200 NE2 801268 OH 801268 OH 801354 OH 801354 OH 801354 OH 801361 OH 801361 OH 8013960H 8013970H 801397 OH

Human Carbonic Anhydrase I-Bicarbonate Complex Table 2 H -bond distances (A)

HCAI 2.6 2.6 3'0 2'8 3.1 2'6 2.8 3'2 2'7 3'0

HCAI-HCO:! 2'7 2.8 2'9 3'3 3'1 2'8 2.9 3'3 2'8 3'1 3.2 3,0 2.8 2.8 2'5 2'8 2'8 3.2 2'9 3,0 2.8 3.2 2'5 3,0 2.8 3'1 2'5 2'5 3'2 3.1

801264 OH 8012690H 8012690H 801396 OH 8012670H 801268 OH 8012720H Gln92 NE2 His94 ND I 8013540H 8014870H Glull7 OE2 Thrl990GI Arg246 N 801264 OH Glull70EI His 119 NE2 HC0522 02 HC052201 801281 OH 801272 OH 801269 OH 8012720H 801361 OH 8014220H 8014870H 8014870H HC0522 03 8014220H 801422 OH 801418 OH

Bicarbonate anion (occupancy 40%) binds to zinc ion at an apical tetrahedral site (site A), replacing the activity linked H20/OH- group in the native HCAI, with one oxygen atom (0-1) at 1-8A, and Zn-C distance of 2-7 A (Figure I). The 0-2 atom of HC03" accepts a H-bond from the -NH group of

in the active site

Thrl99

= 3-2 A;

(dN-0-2

angles C