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R Wolfenden

Publications and source records attributed to R Wolfenden.

At least 55 records · Page 3Linked to original sources
PubMed1989

Binding of pyrimidin-2-one ribonucleoside by cytidine deaminase as the transition-state analogue 3,4-dihydrouridine and the contribution of the 4-hydroxyl group to its binding affinity.

Cytidine deaminase, purified to homogeneity from constitutive mutants of Escherichia coli, was found to bind the competitive inhibitors pyrimidin-2-one ribonucleoside (apparent Ki = 3.6 x 10(-7) M) and 5-fluoropyrimidin-2-one ribonucleoside (apparent Ki = 3.5 x 10(-8) M). Enzyme binding resulted in a change of the lambda max of pyrimidin-2-one ribonucleoside from 303 nm for the free species to 239 nm for the bound species. The value for the bound species was identical with that of an oxygen adduct formed by combination of hydroxide ion with 1,3-dimethyl-2-oxopyrimidinium (239 nm), but lower than that of a sulfur adduct formed by combination of the thiolate anion of N-acetylcysteamine with 1,3-dimethyl-2-oxopyrimidinium (259 nm). The results suggest that pyrimidin-2-one ribonucleoside is bound by cytidine deaminase as an oxygen adduct, probably the covalent hydrate 3,4-dihydrouridine, rather than intact or as an adduct involving a thiol group of the enzyme. In dilute solution at 25 degrees C, the equilibrium constant for formation of a single diastereomer of 3,4-dihydrouridine from pyrimidin-2-one ribonucleoside was estimated as approximately 4.7 x 10(-6), from equilibria of dissociation of water, protonation of 1-methylpyrimidin-2-one, and combination of the 1,3-dimethylpyrimidinium cation with the hydroxide ion.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytidine↗
PubMed1989

Contribution of a single hydroxyl group to transition-state discrimination by adenosine deaminase: evidence for an "entropy trap" mechanism.

Adenosine deaminase was found to bind 6-hydroxy-1,6-dihydropurine ribonucleoside (II), formed by reversible addition of water to purine ribonucleoside (I) in a reaction analogous to formation of a tetrahedral intermediate in substrate deamination, with an apparent Ki value of 3 x 10(-13) M at 20 degrees C. 1,6-Dihydropurine ribonucleoside (IV), synthesized by photolysis of purine ribonucleoside in the presence of NaBH4, exhibited a Ki value of 5.4 x 10-6 M. After correction for differences between the relative free energies of solvation of II and IV, the 6-hydroxyl group of II was estimated to contribute more than 16 kcal to the free energy of binding, approaching the enthalapy of formation of a single hydrogen bond to charged group in the vapor phase. The relatively weak binding of IV and of substrate water suggests that entropic effects, arising from the cooperative action of binding determinants contained within these separate molecules, contribute more than 10 kcal/mol to the free energy of binding of II in which these binding determinants are contained within a single molecule. In free solution, the entropy of reversible hydration of I was evaluated by measuring the temperature dependence of equilibria of protonation of I and of pseudobase formation from I-methylpurinium ribonucleoside as -35 eu, comparable with the entropy of activation for the uncatalyzed hydrolysis of adenosine. In the active site of adenosine deaminase, this thermodynamic obstacle is evidently climbed spontaneously as a result of attractive interactions between the active site and the critical hydroxyl group at the 6-position.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Deaminase↗
PubMed1989

Incomplete factorial search for conditions leading to high quality crystals of Escherichia coli cytidine deaminase complexed to a transition state analog inhibitor.

We have used an incomplete factorial design (Carter, C. W., and Carter, C. W., Jr. (1979) J. Biol. Chem. 254, 12219-12223) to find conditions for growing high quality crystals of Escherichia coli cytidine deaminase (EC 3.5.4.5). Crystals grow at pH 6.0 in hanging or sitting drops with either 1.6 M ammonium sulfate or 2.4-2.5 M sodium phosphate as precipitant. Both conditions produce crystals with identical morphologies and unit cell constants. The space group is P3(1)21 (or its enantiomorph P3(2)21), and the unit cell constants are a = b = 120.3 A, c = 78.4 A. The asymmetric unit is most reasonably one dimer of 66,000 Mr. The crystal size is very dependent on the supersaturation ratio, S = [initial protein concentration]/[equilibrium protein concentration], exhibiting a maximum at S = 7.7. The largest crystals diffract to at least 2.5 A and have a lifetime of 4 to 5 days in the x-ray beam at room temperature. The enzyme in these crystals is complexed with the transition state analog inhibitor 1-(beta-D-ribofuranosyl)-5-fluoropyrimidin-2-one (5-fluoropyrimidin-2-one riboside). We have collected data from parent crystals and from a heavy atom derivative in which the transition state analog is replaced by the active site directed inhibitor 5-(chloromercuri)cytidine.

Crystallization↗
PubMed1989

Major enhancement of the affinity of an enzyme for a transition-state analog by a single hydroxyl group.

The compound 1,6-dihydropurine ribonucleoside, prepared by reduction of nebularine in the presence of ultraviolet light, is bound by adenosine deaminase approximately 10(8)-fold less tightly than 6-hydroxy-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog. This difference in affinities, which is associated with the presence of a single hydroxyl group in the second compound, suggests the degree to which one or a few hydrogen bonds may stabilize the transition state in an enzyme reaction of this type.

Adenosine Deaminase↗
PubMed1989

Transition-state stabilization by adenosine deaminase: 1,6-addition of water to purine ribonucleoside, the enzyme's affinity for 6-hydroxy-1,6-dihydropurine ribonucleoside, and the effective concentration of substrate water at the active site.

Positions of equilibria of highly unfavorable addition reactions, whose products are present at concentrations below the limits of detection, can be determined from equilibria of combination of anionic nucleophiles with quaternized enamines. Applied to the newly prepared 1-methylpurinium ribonucleoside cation, this method yields approximate equilibrium constants of 2 X 10(-9) M-1 for addition of water and 4 X 10(-5) M-1 for addition of N-acetylcysteine to neutral purine ribonucleoside, in dilute aqueous solution. Positions of 13C magnetic resonances and UV absorption maxima of the above complexes and comparison with those of adenosine deaminase complexes strongly suggest that purine ribonucleoside is bound by adenosine deaminase as the 1,6 covalent hydrate, not as a covalently bonded complex formed by addition of a thiol group at the active site. The favorable position of equilibrium of the hydration reaction on the enzyme, together with its extremely unfavorable position in free solution, indicates that the effective activity of substrate water at the active site is in the neighborhood of 10(10) M. The Ki value of the active diastereomer of 6-hydroxy-1,6-dihydropurine ribonucleoside is estimated as 1.6 X 10(-13) M, more than 8 orders of magnitude lower than the apparent dissociation constants of enzyme complexes with the substrate adenosine or the product inosine. The enzyme's remarkable affinity for this hydrated species, which is vanishingly rare in free solution, seems understandable in terms of the hydrate's close resemblance to a hydrated intermediate approaching the transition state in direct water attack on adenosine.

Adenosine Deaminase↗
PubMed1988

Equilibrium of 5,6-hydration of NADH and mechanism of ATP-dependent dehydration.

At equilibrium, water addition to the 5,6 double bond of NADH was observed to favor the hydrate by a factor of approximately 100. Hydration generates two epimers of NADHX (beta-6-hydroxy-1,4,5,6-tetrahydronicotinamide adenine dinucleotide). Only the 6S epimer of the hydrate was found to serve as a true substrate for an ATP-dependent dehydratase from yeast that regenerates NADH. Yet enzymatic conversion of both epimers of the hydrate to NADH was found to proceed essentially to completion in the presence of ATP and dehydratase. This is explained by the observed ability of the epimers to undergo rapid spontaneous equilibration, so that it is unnecessary to postulate a lack of stereospecificity in the dehydratase.

Adenosine Diphosphate Ribose↗
PubMed1988

Influences of solvent on group transfer potentials and biochemical recognition of carbohydrates. Anomalous solvation of the anomeric hydroxyl group.

When carbohydrates in aqueous solution combine with biological receptors, their interactions with the binding site take the place of previous interactions with solvent water. Free energies of binding can therefore be considered to depend on free energies of solvation of the interacting partners before and after complexation. Because carbohydrates contain so many polar substituents, their overall preferences for aqueous surroundings are overwhelming, so that they do not yield readily to analysis of their solvation properties. We therefore decided to eliminate most of the secondary hydroxyl groups and to compare the distribution properties of simple sugar analogs based on tetrahydropyran. The results suggest that compounds containing an anomeric hydroxyl group are less strongly solvated than expected from the properties of simple alcohols. Group transfer potentials of O- and N-glycosides, lactones, and acylals are substantially affected by differences in free energy of solvation between reactants and products. In contrast, equilibria of ring chain tautomerism and covalent hydration of aldoses are almost completely insensitive to the polarity of their surroundings.

Algorithms↗
PubMed1988

Influences of solvent water on protein folding: free energies of solvation of cis and trans peptides are nearly identical.

Peptide bonds interact so strongly with water that even a modest difference between the free energies of solvation of their cis and trans isomers could have a significant bearing on protein structure. However, proton magnetic resonance studies at high dilution in deuteriated solvents show that N-methylformamide exists as the cis isomer to the extent of 8% in water, 10.3% in chloroform, 8.8% in benzene, and 9.2% in cyclohexane. Integrated intensities of proton and carbon resonances show that N-methylacetamide exists as the cis isomer to the extent of only 1.5% in water, not changing much in nonpolar solvents. Quantum mechanical calculations using the 6-31G basis set reproduce these relative abundances with reasonable accuracy and show that there is little difference between the dipole moments of the cis and trans isomers, for either amide. The remarkable insensitivity of cis/trans equilibria to the solvent environment and the heavy preponderance of trans isomers regardless of the polarity of the surroundings (ca. 98.5% for N-methylacetamide, whose properties may resemble those of a typical peptide bond) accord with the overwhelming preference of peptide bonds for the trans configuration that is consistently observed in the three-dimensional structures of globular proteins.

Acetamides↗
PubMed1986

Inhibition of adenosine and thymidylate kinases by bisubstrate analogs.

Potential bisubstrate analogs, in which the 5'-hydroxyl group of adenosine was joined to the phosphoryl group acceptor by polyphosphoryl bridges of varying length (ApnX, where n is the number of phosphoryl groups and X is the nucleoside moiety of the acceptor), were tested as inhibitors of human liver adenosine kinase and of thymidylate kinase from peripheral blast cells of patients with acute myelocytic leukemia. Adenosine kinase was most strongly inhibited by P1,P4-(diadenosine 5')-tetraphosphate (Kd = 30 nM) and P1,P5-(diadenosine 5')-pentaphosphate (Kd = 73 nM). Thymidylate kinase was most strongly inhibited by P1-(adenosine 5')-P5-(thymidine 5')-pentaphosphate (Kd = 120 nM) and by P1(adenosine 5')-P6-(thymidine 5')-hexaphosphate (Kd = 43 nM). In these enzymes, as in adenylate and thymidylate kinases, strongest inhibition was achieved in compounds containing one or two more phosphoryl groups than the substrates combined. These results support the view that nucleoside and nucleotide kinases mediate direct transfer of phosphoryl groups from ATP to acceptors, rather than acting by a double displacement mechanism.

Adenine Nucleotides↗
PubMed1986

Inhibition of thymidine kinase by P1-(adenosine-5')-P5-(thymidine-5')-pentaphosphate.

Potential bisubstrate analogs, with adenosine and thymidine joined at their 5' positions by polyphosphoryl linkages of varying lengths (ApndT, where n = the number of phosphoryl groups), were examined as inhibitors of cytosolic thymidine kinase from blast cells of patients with acute myelocytic leukemia. Ki values were 1.2 microM for Ap3dT, 0.31 microM for Ap4dT, 0.12 microM for Ap5dT, and 0.19 microM for Ap6dT. The best inhibitor of the cytosolic enzyme, Ap5dT, was somewhat less effective as an inhibitor of the mitochondrial enzyme (Ki = 0.50 microM). In addition to their inhibitory modes of binding by the cytosolic enzyme, these compounds were bound at considerably lower concentrations (Kd = 0.029 microM for Ap4dT, 0.0025 microM for Ap5dT, and 0.0027 microM for Ap4dT), in such a way as to protect the cytosolic enzyme from thermal inactivation at 37 degrees C in the absence of substrates.

Adenine Nucleotides↗
PubMed1986

Transition-state stabilization by adenosine deaminase: structural studies of its inhibitory complex with deoxycoformycin.

Experiments with radioactive deoxycoformycin indicate that the inhibitor is released from calf intestinal adenosine deaminase after the enzyme-inhibitor complex is disrupted by denaturation. Experiments with 2H2O and H218O indicate that the enzyme does not catalyze elimination-addition reactions that could have led to reversible covalent derivatization of the enzyme. Ultraviolet difference spectra and the influence of pH on inhibitor binding indicate that deoxycoformycin is bound intact as the neutral species, at a binding site that is less polar than solvent water. The enzyme-inhibitor complex appears to be held together by hydrogen bonds of extraordinary stability (ca. 10 kcal/mol). These results suggest that deamination proceeds by direct water attack, the enzyme acting as a general-base catalyst.

Adenosine Deaminase Inhibitors↗
PubMed1985

Mechanistic implications of the inhibition of peptidases by amino aldehydes and bestatin.

alpha-Amino aldehydes and bestatin are found to be effective inhibitors of a cytosolic dipeptidase (rat testicular peptidase C), and a cytosolic tripeptidase (rat kidney peptidase B, EC 3.4.11.4), as well as cytosolic leucine aminopeptidase (pig kidney peptidase S, EC 3.4.11.1). Aldehyde hydrates and bestatin share a resemblance to intermediates that might be formed during direct attack by water on peptide substrates, affording a possible explanation for their tight binding. Alternatively, inhibitors of both kinds might form derivatives of an active site nucleophile, resembling intermediates in a double-displacement mechanism. Exchange experiments with H218O suggest that bestatin is bound intact by leucine aminopeptidase, lending support to the first of these two mechanisms.

Alanine↗
PubMed1985

Use of secondary isotope effects and varying pH to investigate the mode of binding of inhibitory amino aldehydes by leucine aminopeptidase.

Ki values for leucine aldehyde, a competitive inhibitor of leucine aminopeptidase, vary with pH in a manner compatible with binding of uncharged inhibitor. The pH dependence of kcat/Km suggests likewise that the substrate leucine p-nitroanilide is productively bound as the uncharged species. Comparison of pKa values of the model compounds aminoacetone and aminoacetal indicates that the equilibrium constant for hydration of amino aldehydes is reduced by a factor of about 2 when a proton is lost from the alpha-ammonium group near pH 8. Effects of deuterium substitution at C-1 on equilibrium binding of leucine aldehyde were determined with immobilized enzyme and inhibitors doubly labeled with radioisotopes. The observed isotope effect (KD/KH) is approximately unity, suggesting that leucine aldehyde combines with the enzyme as an oxygen adduct, not as the intact aldehyde.

Deuterium↗
PubMed1983

Waterlogged molecules.

Measurements of vapor pressures over their aqueous solutions indicate that organic compounds show profound differences in hydrophilic character. These differences are of such magnitude as to suggest an important role for changing solvation in determining free energy changes associated with metabolic transformations in water, and in governing structural equilibria of proteins and other large molecules in water. When two or more functional groups are present within the same solute molecule, their combined effects on its free energy of solvation are commonly additive. Striking departures from additivity, observed in certain cases, indicate the existence of special interactions between different parts of a solute molecule and the water that surrounds it. Similar considerations presumably apply to activated intermediates in the interconversion of biological materials.

Chemistry, Organic↗
PubMed1983

Leucinal inhibits brain aminopeptidase activity and potentiates analgesia induced by leu-enkephalen.

L-Leucinal, a transition state analog inhibitor or kidney leucine amino-peptidases, was found to serve as a competitive inhibitor of soluble aminopeptidase activity from rat brain (Ki = 22 microM). Simultaneous intracisternal administration of leucinal and leu-enkephalin produced dose-dependent analgesia in the hot plate test in mice, in concentrations at which neither L-leucinal nor leu-enkephalin alone elicited a significant response. simultaneous administration of leucinal and beta-endorphin also resulted in potentiation of beta-endorphin's analgesic activity. Leucinal did not enhance the antinociceptive response to neurotensin or to [D-ala2]-met-enkephalinamide, which are structurally resistant to the action of brain aminopeptidases.

Aminopeptidases↗
PubMed1982

Inhibition of histone acetylation by N-[2-(S-coenzyme A)acetyl] spermidine amide, a multisubstrate analog.

A multisubstrate analog, formed by joining coenzyme A with spermidine through an acetic acid linkage, serves as a strong inhibitor (Ki less than 10(-8) M) of the acetylation of spermidine and histones by histone acetylase purified from calf thymus. In free solutions, this analog inhibited acetylation of the various nuclear histones to a similar extent. In isolated nuclei, this analog was found to inhibit acetylation of histones H2a and H2b very much more strongly than that of histones H3 and H4.

Acetylation↗
PubMed1982

alpha-aminoaldehydes: transition state analogue inhibitors of leucine aminopeptidase.

L-Leucinal, prepared by enzymatic oxidation of L-leucinol with alcohol dehydrogenase, is found to be a very strong competitive inhibitor of porcine kidney aminopeptidases. For the enzyme from kidney microsomes acting on L-leucine p-nitroanilide (Km = 5.2 x 10(-4) M), for Ki for L-leucinal was 7.6 x 10(-7) M at pH 7.2 and 25 degrees C. For the enzyme from kidney cytosol acting on L-leucine p-nitroanilide (Km = 7.7 x 10(-4) M), Ki for L-leucinal was 6 x 10(-8) M; Ki for glycinal (analogous to glycine derivatives that are poor substrates) was 6.8 x 10(-4) M. In dilute aqueous solution, leucinal exists in unfavorable equilibrium with its covalent hydrate, whose concentration exceeds that of the free aldehyde by a factor of 40. The affinity of the enzyme for the free aldehyde is correspondingly greater than its Ki values would suggest, exceeding the apparent affinity of the substrate by a factor of about 10(6). A comparison of binding affinities suggests that L-leucinal forms an inhibitory complex analogous in structure to unstable intermediates and substrate transformation by leucine aminopeptidase, and strengthens the likelihood that this enzyme may act by a double-displacement mechanism.

Anilides↗