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M L Bender

Publications and source records attributed to M L Bender.

At least 37 records · Page 2Linked to original sources
PubMed1977

Kinetic studies of immobilized alpha-chymotrypsin in apolar solvents.

The mechanism of alpha-chymotrypsin action has been probed by extending studies of native chymotrypsin to immobilized chymotrypsin, where the organic content of the solution can be raised to much higher levels and thus one can explicitly look at the role of water. When one does this, one finds that water only appears in the deacylation reaction. The premise that one can go from native chymotrypsin (souble) to immobilized chymotrypsin (insoluble) has been tested by several criteria. It has been found in many instances that the two are identical: in absolute rate, in pKa. They are, however, not identical to one another in binding, due to differences in diffusion, which is to be expected. Thus, mechanistically immobilized and native chymotrypsin are identical to one another and the use of immobilized chymotrypsin can be used to specify the mechanism even more: it must proceed through two tetrahedral intermediates and two acyl-enzyme intermediates.

Chymotrypsin↗
PubMed1977

Intramolecular general base-catalyzed ester hydrolyses by the imidazolyl group.

Intramolecular general base catalysis by the imidazolyl group was found in the hydrolyses of endo-5-[4;(5')-imidazolyl]-bicyclo[2.2.1]hept-endo-2-yl trans-cinnamate and endo-5-[4'(5')-imidazolyl]bicyclo[2.2.2]oct-endo-2-yl trans-cinnamate in which the imidazolyl and trans-cinnamoyl groups are bound in close proximity to each other by rigid bicyclic rings. The rate constants for the intramolecular general base-catalyzed hydrolyses at 60 degrees are 6.4 X 10(-7) sec-1 for the former and 1.8 X 10(-7) sec-1 for the latter and the deuterium oxide solvent isotope effects are 3.0 for both. On the other hand, no intramolecular catalytic participation of the imidazolyl group was observed in the hydrolyses of the endo-exo isomers, exo- 5-[4'(5')-imidazolyl]bicyclo[2.2.1]hept-endo-2-yl trans-cinnamate and endo-5[4'(5')-imidazolyl]bicyclo[2.2.2]oct-exo-2-yl trans-cinnamate, in which the imidazolyl groups are located far from the trans-cinnamoyl groups. Intramolecular general base-catalyzed hydrolyses by the imidazolyl groups in endo-5[4'(5')-imidazolyl]bicyclo[2.2.1]hept-endo-2-yl trans-cinnamate and endo-5-[4'(5')-imidazolyl]bicyclo[2.2.2]oct-endo-2-yl trans-cinnamate can serve as models of serine esterase-catalyzed hydrolyses.

Catalysis↗
PubMed1977

Model for "charge-relay": acceleration by carboxylate anion in intramolecular general base-catalyzed ester hydrolysis by the imidazolyl group.

The effect of benzoate anion on intramolecular general base-catalyzed ester hydrolysis by the imidazolyl group in endo-5-[4'(5')-imidazolyl]bicyclo[2.2.1]hept-endo-2-yl trans-cinnamate was examined in dioxane/H(2)O solutions. Benzoate anion exhibited a remarkable acceleration of the intramolecular general base-catalyzed hydrolysis of endo-5-[4'(5')-imidazolyl]bicyclo[2.2.1]hept-endo-2-yl trans-cinnamate by the imidazolyl group. The rate of hydrolysis in the presence of the benzoate anion increased with the dioxane mole fraction and was proportional to the concentration of benzoate anion. On the other hand, the rate of hydrolysis of endo-5-[4'(5')-imidazolyl]bicyclo[2.2.1]hept-endo-2-yl trans-cinnamate in the absence of benzoate anion decreased with the dioxane mole fraction. Thus, the ratio of the rate in the presence of benzoate anion to that in the absence of benzoate anion drastically increased with the dioxane mole fraction and attained a 2500-fold rate acceleration at a dioxane mole fraction of 0.42 (the highest experimentally attainable) when the concentration of benzoate anion was 0.5 M. The proposed mechanism involves proton abstraction by the benzoate anion from the imidazolyl group, followed by proton abstraction by the imidazolyl group from H(2)O, resulting in effective general base-catalysis of hydrolysis. The results of the present paper provide support for the "charge-relay" system in serine proteases.

Benzoates↗
PubMed1976

Kinetics of alpha-chymotrypsin dimerization.

A method has been devised which permits the observation of the loss of active sites promoted by aggregation of alpha-chymotrypsin. When alpha-chymotrypsin in unbuffered solution at pH 7 is mixed with buffered proflavin by stopped flow instrumentation to give a final pH of 3.89, a decrease in active sites occurs, as measured by a decrease in enzyme-dye complex. The decrease in the rate of active sites shows a linear dependence on the square of the concentration of active sites remaining at equilibrium. The kinetic data of the reaction have been correlated with equilibrium measurements. Rate constants for formation and dissociation of dimer are 9.45 X 10(3) M(-1)S(-1) and 1.9 S(-1),, respectively. Calculation of Kdis for dimer from rate constants gives a value of 2.01 X 10(-4) M, while direct determination of Kdis gives a value of 1.44 X 10(-4) M.

Binding Sites↗
PubMed1976

Amine-catalyzed hydrolyses of cyclodextrin cinnamates.

Hydrolyses of beta-cyclodextrin cinnamate (betaCDC) and alpha-cyclodextrin cinnamate were catalyzed by amines such as 1,4-diazabicyclo(2.2.2)octane, triethylamine, quinuclidine, piperidine, diisobutylamine, and n-butylamine. The rate constant of hydrolyses of the betaCDC-amine complexes follows the order: 1,4-diazabicyclo(2.2.2)octane > n-butylamine > quinuclidine > piperidine > triethylamine >> diisobutylamine. The ratio of the catalytic rate constant for the betaCDC/1,4-diazabicyclo(2.2.2)octane complex to the spontaneous rate constant for betaCDC is about 6-fold and is almost independent of pH below pH 11.5; but, it then drastically increases with pH above pH 11.5, up to 57-fold at pH 13.6 which is much higher than previous attempts. The pH-rate constant profile and isotope effect with deuterium oxide solvent indicate that 1,4-diazabicyclo(2.2.2)octane, included in betaCDC, assists the catalytic nucleophilic attack by hydroxide ion toward the carbonyl carbon of betaCDC. Acceleration of deacylation of acyl-cyclodextrins, by amines, has made the cyclodextrin-catalyzed hydrolysis of esters an even better model of hydrolytic enzyme reactions than those developed previously.

Journal Article↗
PubMed1975

Acetylation of human serum albumin by p-nitrophenyl acetate.

Human serum albumin reacts very rapidly with p-nitrophenyl acetate (NphOAc). Rapid acetylation of the protein accompanies and largely accounts for the easily observed rapid formation of of p-nitrophenolate ion. One group is acetylated much faster than all others. It appears to be located in a high affinity binding site for small fatty acid anions, to have a pKa of 8.7, and a limiting bimolecular rate of reaction with NphOAc of approximately 3 X 10(4) M-1 sec-1 at alkaline pH values. Rapid reversible binding appears to be a major contributor to the high reaction velocity.

Acetates↗
PubMed1974

Acylation of alpha-chymotrypsin by oxygen and sulfur esters of specific substrates: kinetic evidence for a tetrahedral intermediate.

The acylation step of the alpha-chymotrypsincatalyzed hydrolysis of N-acetyl-L (or DL)-tryptophan p-nitrophenyl, p-nitrothiophenyl, ethyl, and thiolethyl esters has been studied by the stopped-flow technique at 25 degrees . The acylation rate constant, k(2), and the enzyme substrate dissociation constant, K(s), were directly determined at pH 4, 5, and 8. Steady-state kinetics were studied at pH 7. The k(2) values are nearly identical for oxygen esters and their sulfur counterparts, whereas the K(s) value of the ethyl ester is larger by an order of magnitude than those of the other three. The results strongly suggest that oxygen and thiol esters of these specific substrates are hydrolyzed via the same pathway, and furthermore that acylation consists of more than one step, the formation and breakdown of a tetrahedral intermediate, the former being rate-determining. Effects of leaving-group hydrophobicity on k(2) and K(s) are also discussed.

Acetates↗