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Biomedical subjects

R Wolfenden

Publications and source records attributed to R Wolfenden.

At least 73 records · Page 4Linked to original sources
PubMed1981

Affinities of nucleic acid bases for solvent water.

Equilibria of transfer of pyridine and benzene derivatives, from the vapor phase to dilute aqueous solution, are enhanced by the introduction of exocyclic amino and hydroxyl substituents. Much larger increases are associated with the introduction of imino and keto substituents. Purine derivatives exhibit comparable behavior. These observations are discussed in relation to group transfer potentials, the observed affinities of nucleic acid bases for the active sites of proteins, environmental influences on the occurrence of rate tautomers that lead to errors in base pairing, and hypotheses concerning the origins of the genetic code.

Chemical Phenomena↗
PubMed1981

Affinities of amino acid side chains for solvent water.

Equilibria of distribution of amino acid side chains, between their dilute aqueous solutions and the vapor phase at 25 degrees C, have been determined by dynamic vapor pressure measurements. After correction to pH 7, the resulting scale of "hydration potentials", or free energies of transfer from the vapor phase to neutral aqueous solution, spans a range of approximately 22 kcal/mol. The side chain of arginine is much more hydrophilic than those of the other common amino acids, with an equilibrium constant of approximately 10(15) for transfer from the vapor phase to neutral aqueous solution. Hydration potentials are more closely correlated with the relative tendencies of the various amino acids to appear at the surface of globular proteins than had been evident from earlier distribution studies on the free amino acids. Both properties are associated with a pronounced bias in the genetic code.

Amino Acids↗
PubMed1980

Transition state affinity jump chromatography. A double selection method for isolating catalytically active enzymes and other molecules.

A double selection method for isolating active enzyme molecules, using substrate analog affinity chromatography and elution with transition state analogs, is described. To demonstrate the principle, a mixture containing native chymotrypsin and [3H]deoxychymotrypsin, in which the active site serine had been converted to [3H]alanine, was applied to a column containing immobilized D-tryptophan methyl ester. Both forms of chymotrypsin were retained. Catalytically active enzyme was selectively desorbed with the peptide aldehyde chymostatin, leaving catalytically inactive deoxychymotrypsin bound to the substrate analog affinity column. This affinity technique may afford a simple and general method for separating enzymes and other catalysts according to their molecular turnover numbers.

Alanine↗
PubMed1979

Inhibition of ribose-5-phosphate isomerase by 4-phosphoerythronate.

Hoping to exploit the special affinity of enzymes for unstable intermediates in substrate transformation, we have determined the effectiveness of possible analogs of ene-diolate intermediates as inhibitors of spinach ribose-5-phosphate isomerase. 4-Phosphoerythronic acid was found to be a very strong competitive inhibitor, with a Ki value almost 3 orders of magnitude lower than the Km value of ribose 5-phosphate, and very much lower than the Ki value of any other inhibitor that was examined.

Aldose-Ketose Isomerases↗
PubMed1978

Interaction of the peptide bond with solvent water: a vapor phase analysis.

A dynamic technique, using radioactivity as a means of detection, makes it possible to measure the partial pressures of highly polar compounds in dilute aqueous solution. The results can be expressed in terms of the dimensionless distribution coefficient for transfer of a compound from dilute aqueous solution to the vapor phase. For acetic acid this coefficient is 1.1 X 10(-5), for acetamide 7.6 X 10(-8), for N-methylacetamide 4.1 X 10(-8), and for N,N-dimethylacetamide 5.4 X 10(-7). Thus acetamide is much more strongly solvated than the uncharged acetic acid molecule. The results suggest: (1) that the peptide bond represents an extreme among uncharged functional groups in the degree to which it is stabilized by solvent water; (2) that the very great hydrophilic character of the peptide bond may be associated mainly with hydrogen bonding of the solvent to the carbonyl oxygen atom (rather than the N-H group); and (3) that the observed equilibria of biosynthesis and hydrolysis of peptide bonds in aqueous solution are largely determined by differences between reactants and products in their free energies of solvation. It is anticipated that where "bound" water is found in proteins, it will often be found to be associated with peptide bonds, and will tend to be associated with the C-O group rather than with the N-H group.

Acetamides↗
PubMed1977

Influence of substituent ribose on transition state affinity in reactions catalyzed by adenosine deaminase.

Adenosine deaminase from calf intestine hydrolyzes adenine at a limiting rate four orders of magnitude lower than that for adenosine, while Km values for these substrates are about the same (Wolfenden, R., et al. (1969), Biochemistry 8, 2412-2415). Reactivity of 6-substituents, toward nucleophilic displacement, is found to be affected only slightly by removal of ribose as a 9-substituent, in model reactions. Substituent ribose thus appears to stabilize, selectively, the transition state for enzymatic deamination. In contrast with the small influence of substituent ribose on the apparent binding affinity of substrates, removal of substituent ribose from a potential transition state analogue, 1,6-dihydro-6-hydroxy-methylpurine ribonucleoside, results in a lowering of its affinity for the enzyme by several orders of magnitude. The synthesis of the analogue and related compounds is described, and their properties compared with those of other photoadducts and of the naturally occurring inhibitors covidarabine and coformycin. Binding of these inhibitors is found to result in the appearance of ultraviolet-absorbing bands in the neighborhood of 323 nm.

Adenosine Deaminase↗
PubMed1977

Antiproteolytic aldehydes and ketones: substituent and secondary deuterium isotope effects on equilibrium addition of water and other nucleophiles.

Equilibrium constants for hydration of ketones, in dilute D2O solution at 34 degrees C, observed by proton magnetic resonance under conditions of slow exchange, were acetone 0.002, chloroacetone 0.08, 1,3-dichloroacetone 4.17, bromoacetone 0.07, and 1,3-dibromoacetone (an inhibitor of papain) 1.85. Neither acetamidoacetone nor N,N-diacetylaminoacetone showed evidence of appreciable hydration in dilute aqueous solution, nor was any hydrate detectable in solutions of tosylglycine chloromethyl ketone. Substitution of acetaldehyde with acylamido substituents, as in several potent reversible inhibitors of papain, was found to enhance its equilibrium constant for covalent hydration by an order of magnitude; these inhibitors are about 90% hydrated in dilute aqueous solution, and their affinity for proteases may have been underestimated accordingly. The effects of deuterium substitution at C-1 of acetaldehyde, on equilibrium addition of oxygen and sulfur nucleophiles, are substantial and vary with the nature of the nucleophile. These isotope effects may be useful as a mean of distinguishing between alternative structures of complexes formed between enzymes and aldehydes.

Acetaldehyde↗
PubMed1977

Thiohemiacetal formation by inhibitory aldehydes at the active site of papain.

Papain is strongly inhibited by aldehydes resembling carboxylic acids, released by hydrolysis of specific substrates (Westerik, J. O'C., and Wolfenden, R. (1972), J. Biol. Chem. 247, 8195-8197). Inhibitory complexes might involve binding of the aldehyde intact or as a covalent hydrate, or the aldehyde might undergo covalent addition of an active site sulfhydryl group to form a thiohemiacetal derivative. In an attempt to distinguish between these possibilities, benzamidoacetaldehyde-1-d has been synthesized, and its properties compared with those of the undeuterated inhibitor. After correction for differences in hydration, the observed effect on inhibition is found to be compatible with formation of a thiohemiacetal. In keeping with this conclusion, benzamidoethanol (a partial analogue of the covalent hydrate) and benzamide, N-methylbenzamide and N-ethylbenzamide (somewhat similar to the free aldehyde in size and hydrophobic character) are found to exhibit negligible affinity for the active site.

Acetaldehyde↗
PubMed1975

The influence of pH on the interaction of inhibitors with triosephosphate isomerase and determination of the pKa of the active-site carboxyl group.

Ionization effects on the binding of the potential transition state analogues 2-phosphoglycolate and 2-phosphoglycolohydroxamate appear to be attributable to the changing state of ionization of the ligands themselves, therefore it is unnecessary to postulate the additional involvement of an ionizing residue at the active site of triosephosphate isomerase to explain the influence of changing pH on Ki in the neutral range. The binding of the competitive inhibitor inorganic sulfate is insensitive to changing pH in the neutral range. 3-Chloroacetol sulfate, synthesized as an active-site-specific reagent for triosephosphate isomerase, is used to provide an indication of the pKa of the essential carboxyl group of this enzyme. Previously described active-site-specific reagents for the isomerase were phosphate esters, and their changing state of ionization (accompanied by possible changes in their affinity for the active site) may have complicated earlier attempts to determine the pKa of the essential carboxyl group from the pH dependence of the rate of inactivation. Being a strong monoprotic acid, chloroacetol sulfate is better suited to the determination of the pKa of the carboxyl group. Chloroacetol sulfate inactivates triosephosphate isomerase by the selective esterification of the same carboxyl group as that which is esterified by the phosphate esters described earlier. From the pH dependence of the rate of inactivation of yeast triosephosphate isomerase, the apparent pKa of the active-site carboxyl group is estimated as 3.9 +/- 0.1.

Animals↗