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R Wolfenden

Publications and source records attributed to R Wolfenden.

At least 37 records · Page 2Linked to original sources
PubMed1995

Glutamate gamma-semialdehyde as a natural transition state analogue inhibitor of Escherichia coli glucosamine-6-phosphate synthase.

Pyrroline-5-carboxylate, an intermediate in the biosynthesis and degradation of glutamate, proline, and ornithine, acts as a strong reversible inhibitor of glucosamine-6-phosphate synthase, competitive with respect to glutamine. Proton magnetic resonance spectroscopy shows that, under these conditions, pyrroline-5-carboxylate exists in rapid equilibrium with glutamate gamma-semialdehyde (0.05%). The observed variation of Ki with pH is consistent with inhibition by this rare species. Glutamate gamma-semialdehyde is expected to react reversibly with a cysteine residue at the active site, identified by earlier inactivation studies, to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. The apparent Ki value of glutamate gamma-semialdehyde is approximately 3 x 10(-8) M.

Enzyme Inhibitors↗
PubMed1995

Transition-state selectivity for a single hydroxyl group during catalysis by cytidine deaminase.

Cytidine deaminase binds transition-state analog inhibitors approximately 10(7) times more tightly than corresponding 3,4-dihydro analogs containing a proton in place of the 4-hydroxyl group. X-ray crystal structures of complexes with the two matched inhibitors differ only near a "trapped" water molecule in the complex with the 3,4-dihydro analog, where contacts are substantially less favorable than those with the hydroxyl group of the transition-state analog. The hydrogen bond between the hydroxyl group and the Glu 104 carboxylate shortens in that complex, and may become a "low-barrier" hydrogen bond, since at the same time the bond between zinc and the Cys 132 thiolate ligand lengthens. These differences must therefore account for most of the differential binding affinity related to catalysis. Moreover, the trapped water molecule retains some of the binding energy stabilizing the hydroxyl group in the transition-state analog complex. To this extent, the ratio of binding affinities for the two compounds is smaller than the true contribution of the hydroxyl group, a conclusion with significant bearing on interpreting difference free energies derived from substituent effects arising from chemical modification and/or mutagenesis.

Catalysis↗
PubMed1995

Major contribution of a carboxymethyl group to transition-state stabilization by cytidine deaminase: mutation and rescue.

The crystal structure of an inhibitory complex formed between Escherichia coli cytidine deaminase and the transition-state analog 3,4-dihydrouridine indicates the presence of a short H-bond between Glu-104 and the inhibitor. To test the possibility that analogous H-bonds might play a significant role in stabilizing the hydrated substrate in the transition state for deamination, we replaced Glu-104 by alanine. Compared with the wild-type enzyme, the mutant enzyme's affinities for substrate cytidine and product uridine were found to have increased, whereas kcat for deamination of cytidine had been reduced by 8 orders of magnitude. By its presence, the carboxymethyl group of Glu-104 appears to minimize the activation barrier for deamination, not only by stabilizing the altered substrate in the transition state but also by destabilizing the enzyme-substrate and enzyme-product complexes. In the presence of added formate ion, but not in the presence of bulkier carboxylic acids, the low catalytic activity of the mutant enzyme was enhanced substantially.

Alanine↗
PubMed1995

A proficient enzyme.

Orotic acid is decarboxylated with a half-time (t1/2) of 78 million years in neutral aqueous solution at room temperature, as indicated by reactions in quartz tubes at elevated temperatures. Spontaneous hydrolysis of phosphodiester bonds, such as those present in the backbone of DNA, proceeds even more slowly at high temperatures, but the heat of activation is less positive, so that dimethyl phosphate is hydrolyzed with a t1/2 of 130,000 years in neutral solution at room temperature. These values extend the known range of spontaneous rate constants for reactions that are also susceptible to catalysis by enzymes to more than 14 orders of magnitude. Values of the second-order rate constant kcat/Km for the corresponding enzyme reactions are confined to a range of only 600-fold, in contrast. Orotidine 5'-phosphate decarboxylase, an extremely proficient enzyme, enhances the rate of reaction by a factor of 10(17) and is estimated to bind the altered substrate in the transition state with a dissociation constant of less than 5 x 10(-24) M.

Catalysis↗
PubMed1994

On the probability of finding a water molecule in a nonpolar cavity.

Measurements of solubility and vapor pressure indicate that the cost of making a cavity in a nonpolar solvent that is large enough to accommodate a single water molecule is just balanced by the attraction between the water molecule and the walls of the cavity. As a result of this unexpected coincidence, the equilibrium constant for entry of an isolated water molecule from the vapor phase into cyclohexane is almost exactly unity at room temperature. Molecules smaller than water prefer the vapor phase, whereas molecules larger than water prefer cyclohexane. In equilibrium with liquid water, the tendency of single water molecules to enter small nonpolar cavities, such as may be present in mutant proteins, is expected to be vanishingly small.

Cyclohexanes↗
PubMed1994

Mutations affecting transition-state stabilization by residues coordinating zinc at the active site of cytidine deaminase.

Cytidine deaminase from Escherichia coli contains 1 mol of tightly bound zinc per enzyme subunit (Yang, C., Carlow, D., Wolfenden, R., & Short, S.A. (1992) Biochemistry 31, 4168-4174). When the metal liganding residues Cys-129 and Cys-132 were replaced by Ala, and His-102 was replaced by Ala, Asn, or Gln, deaminase activities of cell extracts containing these mutant enzymes were decreased by several orders of magnitude relative to that of the wild-type enzyme. After purification, each mutant protein was found to contain less than 0.2 mol of zinc per enzyme subunit, except mutant H102Q, which contained 1 mol of zinc per subunit. The activity of each mutant enzyme increased in the presence of added zinc but never attained wild-type activity. Mutant H102N was unique in that this protein could be purified as a stable apoenzyme, activated by added zinc, and then inhibited by EDTA. This mutant enzyme bound zinc with an apparent Kd value of 6.0 x 10(-10) M and regained maximal activity in the presence of 1 mol of zinc per subunit. Affinities of the mutant cytidine deaminases for the transition-state analogue, 5-fluoropyrimidin-2-one ribonucleoside (3,4) hydrate, were found to decrease in rough proportion to kcat/Km over a range spanning several orders of magnitude. This variation in catalytic efficiency arose mainly from effects on kcat, indicating the involvement of zinc coordination in the catalytic process rather than in substrate binding.

Binding Sites↗
PubMed1994

Cytidine deaminase. The 2.3 A crystal structure of an enzyme: transition-state analog complex.

We have solved the structure of Escherichia coli cytidine deaminase (CDA) complexed to the transition state analog, 5-fluoroprimidin-2-one riboside. The monomer of the alpha 2 CDA dimer is composed of a small N-terminal alpha-helical domain with no obvious connection to the active sites, and two, larger, core domains. The two core domains have nearly identical tertiary structures and are related by approximate 2-fold symmetry, but lack internal amino acid sequence homology. Comparison of the core domain structure with known structures by sequence homology and structural compatibility searches suggests that the CDA tertiary structure cannot be superimposed on any known protein structure. The two active sites per dimer are formed across the subunit interface. The N-terminal core domain provides a pyrimidine nucleoside and zinc-binding pocket and the structurally homologous C-terminal core domain in the other monomer covers this active-site cleft, completely sequestering the ligand from solvent. The deeply buried zinc-binding site is formed by a novel "topological switch point" at the amino termini of two alpha-helices in consecutive alpha-beta-alpha-beta segments. The transition state analog is bound as a covalent hydrate at C4. The inhibitor hydroxyl oxygen atom interacts both with the zinc atom and the Glu104 carboxylate group, affording high differential affinity for the hydroxyl group relative to a hydrogen atom, in a manner reminiscent of that observed in adenosine deaminase (ADA). Unlike the latter enzyme, the zinc atom is coordinated in a tetrahedral ligand field to two cysteine and one histidine ligands, plus the hydroxyl group. Moreover, the inhibitor stereochemistry is of the opposite hand from that of the corresponding ADA inhibitor at C4(R), but is the same at the hydroxyl group O4(S). A consequence of these stereochemical differences is that in CDA a single conserved carboxylate side-chain, Glu104, can provide all of the necessary proton transfer functions involved in generating the zinc hydroxide nucleophile, and protonating the pyrimidine ring nitrogen atom and leaving amino group. The differences in zinc ligands, ligand-binding stereochemistry, and tertiary structures of CDA and ADA strongly suggest that the common features of transition state stabilization arose by convergent evolution.

Amino Acid Sequence↗
PubMed1993

Hypersensitivity of an enzyme reaction to solvent water.

The hydrolytic activity of calf intestinal adenosine deaminase is reduced sharply, but reversibly, in the presence of added methanol, ethanol, acetonitrile, or dioxane. This decrease in kcat/Km appears to be related to diminished water content in the presence of each of these cosolvents. No agreement between cosolvents is observed if enzyme activity is plotted as a function of viscosity or dielectric constant; nor do these cosolvents act as conventional reversible inhibitors. The Km value of adenosine and the Ki values of a substrate analogue (6-dimethylaminopurine ribonucleoside) and a powerful competitive inhibitor (6-hydroxy-1,6-dihydropurine ribonucleoside) increase with decreasing solvent water content, but kcat is unaffected. Values of 1/Km and 1/Ki increase with roughly the 9th power of the concentration of water and show no sign of approaching a maximum value as the concentration of water approaches 55 M. These results are consistent with an equilibrium between an abundant, inactive, relatively dehydrated form of the enzyme and a rare, relatively hydrated form of the enzyme. Only the hydrated form of the enzyme, containing at least nine more water molecules than the dehydrated form, appears to be capable of binding substrates or competitive inhibitors. Possible physiological consequences of this behavior, in a tissue in which water is transported in large quantities, are considered.

Acetonitriles↗
PubMed1993

Lack of water transport by amino acid side chains or peptides entering a nonpolar environment.

Water has been reported to enter cyclohexane in association with 3-methylindole, a model compound representing the side chain of tryptophan. Entrainment of water would cloud the interpretation of measured partition coefficients as a simple index of hydrophobicity. NMR and isotope-exchange experiments indicate that the reported entrainment of water resulted from unrecognized exchange of 3H from water into the -NH- group of the indole ring. A more detailed analysis shows that no significant amounts of excess water (less than 0.1 molecule/molecule of solute) enter cyclohexane with molecules representing the side chains of tryptophan, phenylalanine, threonine, lysine, or the peptide bond itself.

Amino Acids↗
PubMed1993

Transition-state discrimination by adenosine deaminase from Aspergillus oryzae.

Adenosine deaminase from Aspergillus oryzae resembles mammalian adenosine deaminases in its ability to catalyze the hydrolytic removal of many substituents from C-6, and in the chirality at C-6 of the active isomer of the transition-state-analogue inhibitor 6-hydroxymethyl-1,6-dihydropurine ribonucleoside. The 5'-OH group of adenosine has been found to contribute a factor of 5.10(4) to transition-state stabilization by calf intestinal adenosine deaminase, and crystallographic observations suggest that a zinc-histidine 'bridge' is formed between the 6-OH and the 5'-OH groups of the substrate in the transition state for its deamination. The present paper describes experiments indicating that this bridge is not present during the action of adenosine deaminase from Aspergillus oryzae. We find (1), that the fungal enzyme catalyzes deamination of adenosine and 5'-deoxyadenosine with kcat/Km values that are almost identical; (2), that the Ki value of the transition-state-analogue inhibitor 2'-deoxycoformycin is much higher for the fungal enzyme (2.7.10(-9) M) than for the mammalian enzyme (2.10(-12) M) and (3), that this difference in binding affinities arises mainly from a difference in rates of enzyme-inhibitor association. Thus, the onset of inhibition was markedly slower for the fungal enzyme (kon = 1.3.10(4) M-1 s-1) than for the calf intestinal enzyme (kon = 2.6.10(6) M-1 s-1). Effects of chelating agents and divalent cations suggest that the fungal enzyme, like other deaminases for adenosine and cytidine, contains essential zinc.

Adenosine Deaminase↗
PubMed1993

Are there limits to enzyme-inhibitor binding discrimination? Inferences from the behavior of nucleoside deaminases.

An enzyme can enhance the rate of a reaction only to the extent that it binds the altered substrate in the transition state (S not equal to) more tightly than it binds the substrate in the ground state. Inhibitors that resemble S not equal to can be used to stop an enzyme from working, probe its mechanism of action and obtain exact structural information about intermediates in catalysis. In S not equal to analog inhibitors of adenosine and cytidine deaminases, a single hydroxyl group appears to make extremely large contributions to binding affinity. The magnitude of this contribution becomes even more striking when differences in free energy of solvation by water are taken into account. Other results, obtained by deleting individual binding determinants, indicate the operation of remarkable levels of cooperativity and suggest that if every group is in exactly the right position and is part of an inflexible structure, then a single substituent or H-bond can produce very large increases in binding affinity. Some implications for inhibitor design are considered.

Aminohydrolases↗
PubMed1992

A transition state in pieces: major contributions of entropic effects to ligand binding by adenosine deaminase.

Nebularine undergoes hydration at the active site of adenosine deaminase, in a reaction analogous to a partial reaction in the displacement of ammonia from adenosine by water, to generate an inhibitory complex that captures much of the binding affinity expected of an ideal transition-state analogue. Enzyme affinities of several compounds related to nebularine 1,6-hydrate, and to its stable analog 2'-deoxycoformycin, were compared in an effort to identify the structural origins of strong binding. Binding of the stable transition-state analog inhibitor 2'-deoxycoformycin was rendered 9.8 kcal/mol less favorable by removal of substituent ribose, 9.7 kcal/mol less favorable by inversion of the 8-hydroxyl substituent of the diazepine ring, and 10.0 kcal/mol less favorable by removal of atoms 4-6 of the diazepine ring. Binding of the unstable transition-state analog nebularine hydrate was rendered at least 9.9 kcal/mol less favorable by removal of the 6-hydroxyl group and 10.2 kcal/mol less favorable by removal of atoms 1-3 of the pyrimidine ring. In each case, the enzyme exhibited only modest affinity (Kd greater than or equal to 10(-2) M) for the "missing piece", indicating that incorporation of 2 binding determinants within a single molecule permits an additional 7-12 kcal/mol of intrinsic binding energy to be manifested as observed binding energy. These results are consistent with earlier indications that adenosine deaminase may use 10.5 kcal/mol of the intrinsic free energy of binding of the two substrates to place them in positions appropriate for reaction at the active site, overcoming the unfavorable entropy change of -35 eu for the equilibrium of 1,6-hydration of purine ribonucleoside and reducing the equilibrium constant for attainment of the transition state in deamination of adenosine. Thus, adenosine deaminase may achieve up to 8 orders of magnitude of its catalytic power by converting the nonenzymatic, bimolecular, hydration reaction to a monomolecular reaction at its active site. Several new 6-substituted 1,6-dihydropurine ribonucleosides, prepared by photoaddition of formate and by low-temperature addition of organolithium reagents to a derivative of purine ribonucleoside, exhibited Ki values of 9-1400 microM against adenosine deaminase, in accord with the active site's considerable tolerance of bulky leaving groups in substrates. Inhibition by one diastereomer of 6-carboxy-1,6-dihydropurine ribonucleoside was found to be time-dependent, progressing from a weakly bound to a more strongly bound complex.

Adenosine Deaminase↗
PubMed1992

Cloning and nucleotide sequence of the Escherichia coli cytidine deaminase (ccd) gene.

The structural gene that encodes cytidine deaminase (cdd) in Escherichia coli was cloned from Kohara phage lambda 365 (7F1), and its nucleotide sequence was determined. Plasmids harboring the gene complemented chromosomal cdd mutations, enhanced cytidine deaminase activity in cell extracts, and directed the synthesis of a protein identical in mass and N-terminal amino acid sequence with cytidine deaminase purified from wild-type bacteria. Metal analysis of the purified, plasmid-encoded deaminase indicated a single atom of tightly bound zinc per subunit. Earlier work has shown that bacterial cytidine deaminase and mammalian adenosine deaminase are remarkably alike in their mechanisms of action, in their free energies of interaction with analogue inhibitors resembling tetrahedral intermediates in nucleophilic substitution, and in their ability to discriminate between analogue inhibitors differing by a single hydroxyl group. In contrast to these shared catalytic similarities, the deduced amino acid sequence of E. coli cytidine deaminase (monomer MW 31,540) differs markedly from the mammalian adenosine deaminase sequence suggesting major differences in their tertiary structures. Nevertheless, cytidine deaminase and mammalian plus bacterial adenosine deaminases share a single region (TVHA) of sequence identity that is tentatively identified as part of the cytidine deaminase active site.

Amino Acid Sequence↗
PubMed1991

Lyase activity of nucleoside 2-deoxyribosyltransferase: transient generation of ribal and its use in the synthesis of 2'-deoxynucleosides.

In the absence of acceptors nucleoside 2-deoxyribosyltransferase catalyzes the slow hydrolysis of 2'-deoxynucleosides. During this hydrolytic reaction, D-ribal (1,4-anhydro-2-deoxy-D-erythro-pent-1-enitol), a glycal of ribose hitherto encountered only as a reagent in organic synthesis, is generated spontaneously, disappearing later as 2'-deoxynucleoside hydrolysis approaches completion. Nucleoside 2-deoxyribosyltransferase is found to catalyze the hydration of D-ribal in the absence of nucleic acid bases and the synthesis of deoxyribonucleosides from ribal in their presence, affording a new method for the preparation of 2'-deoxyribonucleosides. The stereochemistry of nucleoside formation from ribal supports the intervention of deoxyribosyl-enzyme intermediate. The equilibrium constant for the covalent hydration of ribal is found to be approximately 65.

Deoxy Sugars↗
PubMed1991

Analogues of intermediates in the action of pig kidney prolidase.

Dicarboxylic acids, resembling the collected substrates for the reverse peptide bond forming reaction, were bound several orders of magnitude more tightly than substrates, products, or previously known competitive inhibitors of reactions catalyzed by pig kidney prolidase (EC 3.4.13.9), a dipeptidase that cleaves peptide bonds to the nitrogen atom of proline. Other inhibitors containing a phosphoryl or phosphonyl group in addition to a carboxyl substituent were bound even more tightly, in a manner consistent with their possible resemblance to tetrahedral intermediates in substrate hydrolysis. These included several analogues of phosphoenol pyruvate, of which the most potent was (Z)-3-bromophosphoenolpyruvate (Ki = 4.6 x 10(-9) M). Ki values were found to vary with changing pH in a manner consistent with displacement of a hydroxide ion from the active site.

Animals↗
PubMed1990

Orotidine-5'-monophosphate decarboxylase catalysis: kinetic isotope effects and the state of hybridization of a bound transition-state analogue.

The enzymatic decarboxylation of orotidine 5'-monophosphate may proceed by an addition-elimination mechanism involving a covalently bound intermediate or by elimination of CO2 to generate a nitrogen ylide. In an attempt to distinguish between these two alternatives, 1-(phosphoribosyl)barbituric acid was synthesized with 13C at the 5-position. Interaction of this potential transition-state analogue inhibitor with yeast orotidine-5'-monophosphate decarboxylase resulted in a small (0.6 ppm) downfield displacement of the C-5 resonance, indicating no rehybridization of the kind that might have been expected to accompany 5,6-addition of an enzyme nucleophile. When the substrate orotidine 5'-monophosphate was synthesized with deuterium at C-5, no significant change in kcat (H/D = 0.99 +/- 0.06) or kcat/KM (H/D = 1.00 +/- 0.06) was found to result, suggesting that C-5 does not undergo significant changes in geometry before or during the step that determines the rate of the catalytic process. These results are consistent with a nitrogen ylide mechanism and offer no support for the intervention of covalently bound intermediates in the catalytic process.

Carboxy-Lyases↗