Biomedical subjects

R Wolfenden

Publications and source records attributed to R Wolfenden.

At least 19 recordsLinked to original sources

PubMed2001

Site-bound water and the shortcomings of a less than perfect transition state analogue.

Kinetic measurements have shown that substantial enthalpy changes accompany substrate binding by cytidine deaminase, increasing markedly as the reaction proceeds from the ground state (1/K(m), DeltaH = -13 kcal/mol) to the transition state (1/K(tx), DeltaH = -20 kcal/mol) [Snider, M. J., et al. (2000) Biochemistry 39, 9746-9753]. In the present work, we determined the thermodynamic changes associated with the equilibrium binding of inhibitors by cytidine deaminase by isothermal titration calorimetry and van't Hoff analysis of the temperature dependence of their inhibition constants. The results indicate that the binding of the transition state analogue 3,4-dihydrouridine DeltaH = -21 kcal/mol), like that of the transition state itself (DeltaH = -20 kcal/mol), is associated with a large favorable change in enthalpy. The significantly smaller enthalpy change that accompanies the binding of 3,4-dihydrozebularine (DeltaH = -10 kcal/mol), an analogue of 3,4-dihydrouridine in which a hydrogen atom replaces this inhibitor's 4-OH group, is consistent with the view that polar interactions with the substrate at the site of its chemical transformation play a critical role in reducing the enthalpy of activation for substrate hydrolysis. The entropic shortcomings of 3,4-dihydrouridine, in capturing all of the free energy involved in binding the actual transition state, may arise from its inability to displace a water molecule that occupies the binding site normally occupied by product ammonia.

Ammonia↗

PubMed2001

Role of enzyme-ribofuranosyl contacts in the ground state and transition state for orotidine 5'-phosphate decarboxylase: a role for substrate destabilization?

The crystal structure of yeast orotidine 5'-monophosphate decarboxylase (ODCase) complexed with the inhibitor 6-hydroxyuridine 5'-phosphate (BMP) reveals the presence of a series of strong interactions between enzyme residues and functional groups of this ligand. Enzyme contacts with the phosphoribofuranosyl moiety of orotidine 5'-phosphate (OMP) have been shown to contribute at least 16.6 kcal/mol of intrinsic binding free energy to the stabilization of the transition state for the reaction catalyzed by yeast ODCase. In addition to these enzyme-ligand contacts, active site residues contributed by both subunits of the dimeric enzyme are positioned to form hydrogen bonds with the 2'- and 3'-OH groups of the ligand's ribosyl moiety. These involve Thr-100 of one subunit and Asp-37 of the opposite subunit, respectively. To evaluate the contributions of these ribofuranosyl contacts to ground state and transition state stabilization, Thr-100 and Asp-37 were each mutated to alanine. Elimination of the enzyme's capacity to contact individual ribosyl OH groups reduced the k(cat)/K(m) value of the T100A enzyme by 60-fold and that of the D37A enzyme by 300-fold. Removal of the 2'-OH group from the substrate OMP decreased the binding affinity by less than a factor of 10, but decreased k(cat) by more that 2 orders of magnitude. Upon removal of the complementary hydroxymethyl group from the enzyme, little further reduction in k(cat)/K(m) for 2'-deoxyOMP was observed. To assess the contribution made by contacts involving both ribosyl hydroxyl groups at once, the ability of the D37A mutant enzyme to decarboxylate 2'-deoxyOMP was measured. The value of k(cat)/K(m) for this enzyme-substrate pair was 170 M(-1) s(-1), representing a decrease of more than 7.6 kcal/mol of binding free energy in the transition state. To the extent that electrostatic repulsion in the ground state can be tested by these simple alterations, the results do not lend obvious support to the view that electrostatic destabilization in the ground state enzyme-substrate complex plays a major role in catalysis.

Alanine↗

PubMed2001

Dissecting a charged network at the active site of orotidine-5'-phosphate decarboxylase.

The crystal structure of yeast orotidine-5'-phosphate decarboxylase in complex with the postulated transition state analog, 6-hydroxyuridine-5'-phosphate, reveals contacts between this inhibitor and a novel quartet of charged residues (Lys-59, Asp-91, Lys-93, and Asp-96) within the active site. The structure also suggests a possible interaction between O2 of the 6-hydroxyuridine-5'-phosphate pyrimidine ring and Gln-215. Here we report the results of mutagenesis of each of the charged active site residues and Gln-215. The activities of the Q215A and wild-type enzymes were equal indicating that any interactions between this residue and the pyrimidine ring are dispensable for efficient decarboxylation. For the D91A and K93A mutant enzymes, activity was reduced by more than 5 orders of magnitude and substrate binding could not be detected by isothermal calorimetry. For the D96A mutant enzyme, k(cat) was reduced by more than 5 orders of magnitude, and isothermal calorimetry indicated an 11-fold decrease in the affinity of this enzyme for the substrate in the ground state. For the K59A enzyme, k(cat) was reduced by a factor of 130, and K(m) had increased by a factor of 900. These results indicate that the integrity of the network of charged residues is essential for transition state stabilization.

Binding Sites↗

PubMed2001

The depth of chemical time and the power of enzymes as catalysts.

The fastest known reactions include reactions catalyzed by enzymes, but the rate enhancements that enzymes produce had not been fully appreciated until recently. In the absence of enzymes, these same reactions are among the slowest that have ever been measured, some with half-times approaching the age of the Earth. This difference provides a measure of the proficiencies of enzymes as catalysts and their relative susceptibilities to inhibition by transition-state analogue inhibitors. Thermodynamic comparisons between spontaneous and enzyme-catalyzed reactions, coupled with structural information, suggest that in addition to electrostatic and H-bonding interactions, the liberation of water molecules from an enzyme's active site into bulk solvent sometimes plays a prominent role in determining the relative binding affinities of the altered substrate in the ground state and transition state. These comparisons also indicate a high level of synergism in the action of binding determinants of both the substrate and the enzyme, that are not directly involved in the chemical transformation of the substrate but contribute to the rate of its transformation at an enzyme's active site.

Catalysis↗

PubMed2000

Temperature effects on the catalytic efficiency, rate enhancement, and transition state affinity of cytidine deaminase, and the thermodynamic consequences for catalysis of removing a substrate "anchor".

To obtain a clearer understanding of the forces involved in transition state stabilization by Escherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substrate binding in the ground state and transition state for substrate hydrolysis. Viscosity studies indicate that the action of cytidine deaminase is not diffusion-limited. Thus, K(m) appears to be a true dissociation constant, and k(cat) describes the chemical reaction of the ES complex, not product release. Enzyme-substrate association is accompanied by a loss of entropy and a somewhat greater release of enthalpy. As the ES complex proceeds to the transition state (ES), there is little further change in entropy, but heat is taken up that almost matches the heat that was released with ES formation. As a result, k(cat)/K(m) (describing the overall conversion of the free substrate to ES is almost invariant with changing temperature. The free energy barrier for the enzyme-catalyzed reaction (k(cat)/K(m)) is much lower than that for the spontaneous reaction (k(non)) (DeltaDeltaG = -21.8 kcal/mol at 25 degrees C). This difference, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (S), is almost entirely enthalpic in origin (DeltaDeltaH = -20.2 kcal/mol), compatible with the formation of hydrogen bonds that stabilize the ES complex. Thus, the transition state affinity of cytidine deaminase increases rapidly with decreasing temperature. When a hydrogen bond between Glu-91 and the 3'-hydroxyl moiety of cytidine is disrupted by truncation of either group, k(cat)/K(m) and transition state affinity are each reduced by a factor of 10(4). This effect of mutation is entirely enthalpic in origin (DeltaDeltaH approximately 7.9 kcal/mol), somewhat offset by a favorable change in the entropy of transition state binding. This increase in entropy is attributed to a loss of constraints on the relative motions of the activated substrate within the ES complex. In an Appendix, some objections to the conventional scheme for transition state binding are discussed.

Catalysis↗

PubMed2000

Contribution of enzyme-phosphoribosyl contacts to catalysis by orotidine 5'-phosphate decarboxylase.

The crystal structure of the complex formed between recombinant yeast orotidine 5'-phosphate decarboxylase and the competitive inhibitor 6-hydroxyuridine 5'-phosphate reveals the presence of four hydrogen bonds between active site residues Tyr-217 and Arg-235 and the phosphoryl group of this inhibitor. When Tyr-217 and Arg-235 are individually mutated to alanine, values of k(cat)/K(m) are reduced by factors of 3000- and 7300-fold, respectively. In the Y217A/R235A double mutant, activity is reduced more than 10(7)-fold. Experiments with highly enriched [(14)C]orotic acid show that when ribose 5'-phosphate is deleted from substrate orotidine 5'-phosphate, k(cat)/K(m) is reduced by more than 12 orders of magnitude, from 6.3 x 10(7) M(-1) s(-1) for OMP to less than 2.5 x 10(-5) M(-1) s(-1) for orotic acid. Activity toward orotate is not "rescued" by 1 M inorganic phosphate. The K(i) value of ribose 5'-phosphate, representing the part of the natural substrate that is absent in orotic acid, is 8.1 x 10(-5) M. Thus, the effective concentration of the 5'-phosphoribosyl group, in stabilizing the transition state for enzymatic decarboxylation of OMP, is estimated to be >2 x 10(8) M, representing one of the largest connectivity effects that has been reported for an enzyme reaction.

Binding, Competitive↗

PubMed2000

Anatomy of a proficient enzyme: the structure of orotidine 5'-monophosphate decarboxylase in the presence and absence of a potential transition state analog.

Orotidine 5'-phosphate decarboxylase produces the largest rate enhancement that has been reported for any enzyme. The crystal structure of the recombinant Saccharomyces cerevisiae enzyme has been determined in the absence and presence of the proposed transition state analog 6-hydroxyuridine 5'-phosphate, at a resolution of 2.1 A and 2.4 A, respectively. Orotidine 5'-phosphate decarboxylase folds as a TIM-barrel with the ligand binding site near the open end of the barrel. The binding of 6-hydroxyuridine 5'-phosphate is accompanied by protein loop movements that envelop the ligand almost completely, forming numerous favorable interactions with the phosphoryl group, the ribofuranosyl group, and the pyrimidine ring. Lysine-93 appears to be anchored in such a way as to optimize electrostatic interactions with developing negative charge at C-6 of the pyrimidine ring, and to donate the proton that replaces the carboxylate group at C-6 of the product. In addition, H-bonds from the active site to O-2 and O-4 help to delocalize negative charge in the transition state. Interactions between the enzyme and the phosphoribosyl group anchor the pyrimidine within the active site, helping to explain the phosphoribosyl group's remarkably large contribution to catalysis despite its distance from the site of decarboxylation.

Amino Acid Sequence↗

PubMed1999

Cytidine deaminases from B. subtilis and E. coli: compensating effects of changing zinc coordination and quaternary structure.

Cytidine deaminase from E. coli is a dimer of identical subunits (M(r) = 31 540), each containing a single zinc atom. Cytidine deaminase from B. subtilis is a tetramer of identical subunits (M(r) = 14 800). After purification from an overexpressing strain, the enzyme from B. subtilis is found to contain a single atom of zinc per enzyme subunit by flame atomic absorption spectroscopy. Fluorescence titration indicates that each of the four subunits contains a binding site for the transition state analogue inhibitor 5-fluoro-3,4-dihydrouridine. A region of amino acid sequence homology, containing residues that are involved in zinc coordination in the enzyme from E. coli, strongly suggests that in the enzyme from B. subtilis, zinc is coordinated by the thiolate side chains of three cysteine residues (Cys-53, Cys-86, and Cys-89) [Song, B. H., and Neuhard, J. (1989) Mol. Gen. Genet. 216, 462-468]. This pattern of zinc coordination appears to be novel for a hydrolytic enzyme, and might be expected to reduce the reactivity of the active site substantially compared with that of the enzyme from E. coli (His-102, Cys-129, and Cys-132). Instead, the B. subtilis and E. coli enzymes are found to be similar in their activities, and also in their relative binding affinities for a series of structurally related inhibitors with binding affinities that span a range of 6 orders of magnitude. In addition, the apparent pK(a) value of the active site is shifted upward by less than 1 unit. Sequence alignments, together with model building, suggest one possible mechanism of compensation.

Amino Acid Sequence↗

PubMed1999

Activity of yeast orotidine-5'-phosphate decarboxylase in the absence of metals.

Yeast orotidine-5'-phosphate decarboxylase was recently shown to contain zinc and to be inhibited by zinc-complexing agents. When the gene for the yeast enzyme was expressed in Escherichia coli, the gene product was devoid of metal atoms but exhibited a specific activity and molecular mass similar to those of the enzyme obtained directly from yeast. This invalidates the hypothesis that zinc is involved in substrate decarboxylation. The zinc-free enzyme undergoes thermal inactivation at a somewhat lower temperature than does the zinc-containing enzyme isolated from yeast.

Escherichia coli↗

PubMed1999

Conformational aspects of inhibitor design: enzyme-substrate interactions in the transition state.

The theory of absolute reaction rates suggests that enzymes, like other catalysts, can enhance the rate of a reaction only to the extent that they bind the altered substrate in the transition state (S++) more tightly than they bind the substrate in the ground state (S). ES dissociation constants commonly fall in the physiological range, but recent kinetic studies indicate that formal ES++ dissociation constants of less than 10(-20) M are achieved by enzymes of several classes. Studies with stable analogues suggest that these remarkable powers of discrimination involve a tendency of the enzyme to close around S++ in such a way as to maximize binding contacts; that several parts of the substrate contribute to S++ binding; and that their contributions to binding affinity can be strongly synergistic.

Drug Design↗

PubMed1998

Substrate connectivity effects in the transition state for cytidine deaminase.

The binding properties of substrates and competitive inhibitors of Escherichia coli cytidine deaminase are compared with those of the fragments obtained by cutting these ligands at several positions including the glycosidic bond. In contrast with the normal substrate cytidine (kcat/Km = 2.6 x 10(6) M-1 s-1), cytosine is found to serve as an extremely slow substrate (kcat/Km = 1.8 x 10(-3) M-1 s-1), despite the ability of cytosine to enter any active site that can accommodate the normal substrate cytidine. Spontaneous nonenzymatic deamination proceeds at similar rates for cytosine and cytidine at pH 7 and 25 degrees C, indicating that substituent ribose exerts little effect on the intrinsic reactivity of cytidine in solution. Dividing knon by kcat/Km, the maximal Kd value of the enzyme's complex with the altered substrate in the transition state is estimated as 6.1 x 10(-8) M for cytosine, very much higher than the value (1.2 x 10(-16) M) estimated for cytidine. The Kd value of ribofuranose, the missing substituent, is roughly 1.8 x 10(-2) M, as indicated by the Ki values of D-ribose and 1-methyl-D-ribofuranoside as competitive inhibitors. Thus, the free energy of binding of the altered substrate in the transition state is 9.5 kcal/mol more favorable for the whole molecule cytidine than for the sum of those of its parts, cytosine plus ribofuranose. As a separate molecule, however, ribose shows no detectable effect on the enzyme's activity on cytosine. Connectivity effects of similar magnitude are indicated by the equilibrium binding affinities of inhibitors. Thus, the Ki value of the transition state analogue inhibitor zebularine hydrate (1.2 x 10(-12) M) is very much lower than the combined affinities of N-ribofuranosylurea (1.6 x 10(-4) M) and allyl alcohol (0.14 M), indicating that the glycoside bond, by its presence, exerts a connectivity effect of 9.9 kcal/mol on the observed free energy of binding.

Binding Sites↗

PubMed1998

Hydrophobicities of the nucleic acid bases: distribution coefficients from water to cyclohexane.

To establish an experimental scale of hydrophobicities for the nucleic acid bases, comparable with a scale developed earlier for amino acid side-chains, these bases and their parent compounds (purine and pyrimidin-2-one) were converted to n-butylated and tetrahydrofurylated derivatives that are appreciably soluble in cyclohexane, a truly non-polar solvent that dissolves negligible water at saturation. Distribution measurements between neutral aqueous solution and cyclohexane, at varying solute concentrations, showed no evidence of self-association of the solute in either solvent, and the possibility of specific entrainment of water by solutes entering cyclohexane was ruled out by the results of experiments with tritiated water. In both the n-butyl and tetrahydrofuryl series, the bases span a range of approximately 5.3 kcal mol-1 in their free energies of transfer from water to cyclohexane, and are arranged in the following rank, in order of decreasing hydrophobicity: purine>thymine>adenine>uracil>pyrimidin-2-one>hypoxanthine>/=cytosine >/=guanine. In both series of pyrimidin-2-ones, hydrophobicity decreases with introduction of an amino substituent, but addition of an exocyclic keto group results in a modest enhancement of hydrophobicity; and free energies of transfer are relatively insensitive to the position of N-alkyl substitution. In both series of purines, hydrophobicity decreases with the introduction of exocyclic amino and keto groups, the keto group having the greater effect; and free energies of transfer vary substantially depending on the position of N-alkyl substitution. Several additional compounds were examined to test recent predictions based on SM5.4/A, a quantum mechanical self-consistent-field solvation model; and that model was found to yield values in reasonable agreement with the experimental results.

Adenine↗

PubMed1998

Complementary truncations of a hydrogen bond to ribose involved in transition-state stabilization by cytidine deaminase.

The crystal structure of the complex formed between Escherichia coli cytidine deaminase and the transition-state analogue inhibitor 3,4-dihydrouridine [Betts, L., Xiang, S., Short, S. A., Wolfenden, R., & Carter, C. W. (1994) J. Mol. Biol. 235, 635] shows the presence of an H-bond between Glu-91 and the 3'-OH group of substituent ribose, a part of the substrate that is not directly involved in its chemical transformation. To test the contribution of this interaction to transition-state stabilization, Glu-91 was converted to alanine. The mutant enzyme is very much less active than the wild-type enzyme, with a 500-fold increase in Km and a 32-fold reduction in kcat using cytidine as substrate. No change in secondary structure is evident in the circular dichroic spectrum. As measured by kcat/Km, Glu-91 thus appears to stabilize the transition state for cytidine deamination by an overall factor of 1.7 x 10(4), equivalent to -5.8 kcal/mol in free energy. To test the contribution of this interaction in the opposite sense, the 3'-OH group of the substrate was replaced by a hydrogen atom. Comparing 3'-deoxycytidine with cytidine, the native enzyme shows a 17-fold increase in Km and a 400-fold decrease in kcat, indicating that the 3'-hydroxyl group of cytidine stabilizes the transition state for deamination by an overall factor of 6.3 x 10(3), equivalent to -5.2 kcal/mol in free energy, as measured by kcat/Km. After one binding partner has been removed, however, the effect of removing the remaining partner is relatively slight. For the mutant enzyme E91A, removal of the 3'-hydroxyl group from substrate cytidine reduces kcat/Km by a factor of only 3. Complete removal of substituent ribose reduces the wild-type enzyme's kcat/Km by a factor of more than 10(8); thus, substituent ribose, although distant from the site of chemical transformation of the substrate, contributes at least 11 kcal to the free energy of stabilization of the transition state for cytidine deamination, matching the apparent contribution to transition state binding made by the 4-OH group of the pyrimidine ring, which is at the site of substrate transformation [Frick, L., Yang, C., Marquez, V. E., & Wolfenden, R. (1989) Biochemistry 28, 9423].

Amino Acid Substitution↗

PubMed1997

The structure of the cytidine deaminase-product complex provides evidence for efficient proton transfer and ground-state destabilization.

Crystal structures of the cytidine deaminase-uridine product complex prepared either by cocrystallizing enzyme with uridine or by diffusing cytidine into ligand-free crystals show that the product binds as a 4-ketopyrimidine. They reveal four additional features of the catalytic process. (1) A water molecule bound to a site previously observed to bind the incoming 4-NH2 group represents the site for the leaving ammonia molecule. The conserved Pro 128 accommodates both moieties by orienting the carbonyl group of the previous residue. (2) The Glu 104 carboxylate group rotates from its hydrogen bond to the O4 hydroxyl group in transition-state analog complexes, forming a new hydrogen bond to the leaving group moiety. Thus, after stabilizing the hydroxyl group in the transition state, Glu 104 transfers a proton from that group to the leaving amino group, promoting enol-to-keto isomerization of the product. (3) Difference Fourier comparisons with transition-state complexes indicate that the pyrimidine ring rotates toward the zinc by approximately 10 degrees. The active site thus "pulls" the ring and 4-NH2 group in opposite directions during catalysis. To preserve coplanarity of the 4-keto group with the pyrimidine ring, the N1-C1' glycosidic bond bends by approximately 19 degrees out of the ring plane. This distortion may "spring-load" the product complex and promote dissociation. Failure to recognize a similar distortion could explain an earlier crystallographic interpretation of the adenosine deaminase-inosine complex [Wilson, D. K., & Quiocho, F. A. (1994) Nat. Struct. Biol. 1, 691-694]. (4) The Zn-Sgamma132 bond, which lengthens in transition-state complexes, shortens as the O4 atom returns to a state of lower negative charge in the planar product, consistent with our previous proposal that this bond buffers the zinc bond valence, compensating buildup of negative charge on the oxygen nucleophile during catalysis.

Binding Sites↗

PubMed1997

Mandelate racemase in pieces: effective concentrations of enzyme functional groups in the transition state.

Mandelate racemase (EC 5.1.2.2) catalyzes the abstraction of a proton from a carbon atom adjacent to a carboxylate function, a reaction which is kinetically and thermodynamically unfavorable. Proton NMR spectroscopy and polarimetry were used to measure the rates of deuterium incorporation into the alpha-position of mandelate and the racemization of (R)-mandelate, after samples had been incubated at elevated temperatures. Using an Arrhenius plot, the value of the free energy of activation for racemization and deuterium exchange was calculated to be 34.6 (+/-0.9) kcal/mol under neutral conditions, at 25 degrees C. This result indicates that mandelate racemase produces a remarkable rate enhancement [(1.7 x 10(15))-fold], and a level of transition state affinity (Ktx = 2 x 10(-19) M), that surpasses the levels achieved by most enzymes. Methylamine, imidazole, and acetate catalyzed the nonenzymatic hydrogen-deuterium exchange reaction at 170 degrees C, and the values of the second-order rate constants are 2.8 (+/-0.2) x 10(-5), 13.4 (+/-0.7) x 10(-5), and < or = 4 (+/-1) x 10(-7) M-1 s-1, respectively. By comparing wild-type mandelate racemase's proficiency as a catalyst with the proficiencies of these small molecules which correspond to the missing pieces in the variant enzymes Lys 166 Arg [Kallarakal. A. T., et al. (1995) Biochemistry 34, 2788-2797], His297Asn [Landro, J. A., et al. (1991) Biochemistry 30, 9274-9281], and Glu317Gln [Mitra, B., et al. (1995) Biochemistry 34, 2777-2787], we estimate the effective concentrations of the catalytic side chains of Lys 166, His 297, and Glu 317 as > or = 622, > or = 3 x 10(3), and > or = 3 x 10(5) M, respectively, in the native protein. These observations support the view that general acid-general base catalysis, inefficient in simple model systems, becomes an efficient mode of catalysis when structural complementarity between an enzyme and its substrate is optimized in the transition state.

Binding Sites↗

PubMed1996

Enzyme-substrate complexes of adenosine and cytidine deaminases: absence of accumulation of water adducts.

Adenosine deaminase has been reported to bind the product inosine (the substrate for the reverse reaction) as inosine 1,6-hydrate considered similar in structure to the transition state for adenosine deamination (Wilson & Quiocho, 1994) Accumulation on the enzyme of inosine 1,6-hydrate would be surprising, because this compound is an actual intermediate, probably approaching the transition state, in oxygen exchange between water and the C==O group of inosine, a reaction previously shown to be catalyzed by adenosine deaminase (Wolfenden & Kirsch, 1968). The equilibrium constant for conversion of ES to ES*, in the oxygen exchange reaction, is less than 10-12. To investigate the structure of enzyme-bound inosine in a different way, we labeled deoxyinosine with 13C, excepting an upfield shift of 70-110 ppm if significant rehybridization to sp3 had occurred at the carbonyl group. Instead, the results show a very small shift (1.3 ppm), indicating that C-6 of 2'-deoxyinosine retains its sp2 hybridization after binding by calf intestinal adenosine deaminase. In a separate series of experiments, [4,5-13C]-2'-deoxyuridine was synthesized and found to retain its sp2 hybridization at C-4, after binding by Escherichia coli cytidine deaminase, an enzyme that catalyzes 18O exchange from water into uridine. These findings are consistent with the general expectation, based on the unfavorable equilibrium of activation of enzyme-bound substrates, that enzymes should not accumulate appreciable concentrations of intermediates whose free energies approach that of the transition state in substrate transformation.

Adenosine↗

PubMed1996

Cytidine deaminase complexed to 3-deazacytidine: a "valence buffer" in zinc enzyme catalysis.

The cytidine deaminase substrate analog inhibitor 3-deazacytidine binds with its 4-amino group inserted into a site previously identified as a probable binding site for the leaving ammonia group. Binding to this site shifts the pyrimidine ring significantly further from the activated water molecule than the position it occupies in either of two complexes with compounds capable of hydrogen bonding at the 3-position of the ring [Xiang et al. (1995) Biochemistry 34, 4516-4523]. Difference Fourier maps between the deazacytidine, dihydrozebularine, and zebularine--hydrate inhibitor complexes suggest that the ring itself moves successively toward the activated water, leaving the amino group behind in this site as the substrate complex approaches the transition state. They also reveal systematic changes in a single zinc-sulfur bond distance. These correlate with chemical changes expected as the substrate approaches the tetrahedral transition state, in which the zinc-activated hydroxyl group develops maximal negative charge and forms a short hydrogen bond to the neighboring carboxylate group of Glu 104. Empirical bond valence relationships suggest that the Zn-S gamma 132 bond functions throughout the reaction as a "valence buffer" that accommodates changing negative charge on the hydroxyl group. Similar structural features in alcohol dehydrogenase suggest that analogous mechanisms may be a general feature of catalysis by zinc enzymes.

Catalysis↗

PubMed1996

Role of glutamate-104 in generating a transition state analogue inhibitor at the active site of cytidine deaminase.

The 19F-NMR resonance of 5-[19F]fluoropyrimidin-2-one ribonucleoside moves upfield when it is bound by wild-type cytidine deaminase from Escherichia coli, in agreement with UV and X-ray spectroscopic indications that this inhibitor is bound as the rate 3,4-hydrated species 5-fluoro-3,4-dihydrouridine, a transition state analogue inhibitor resembling an intermediate in direct water attack on 5-fluorocytidine. Comparison of pKa values of model compounds indicates that the equilibrium constant for 3,4-hydration of this inhibitor in free solution is 3.5 x 10(-4) M, so that the corrected dissociation constant of 5-fluoro-3,4-dihydrouridine from the wild-type enzyme is 3.9 x 10(-11) M. Very different behavior is observed for a mutant enzyme in which alanine replaces Glu-104 at the active site, and kcat has been reduced by a factor of 10(8). 5-[19F]Fluoropyrimidin-2-one ribonucleoside is strongly fluorescent, making it possible to observe that the mutant enzyme binds this inhibitor even more tightly (Kd = 4.4 x 10(-8) M) than does the native enzyme (Kd = 1.1 x 10(-7) M). 19F-NMR indicates, however, that the E104A mutant enzyme binds the inhibitor without modification, in a form that resembles the substrate in the ground state. These results are consistent with a major role for Glu-104, not only in stabilizing the ES++ complex in the transition state, but also in destabilizing the ES complex in the ground state.

Binding Sites↗