ENZYME MODELS BASED ON BORONIC ACIDS AND ON A MONOCLONAL ANTIBODY.
Item
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Title
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ENZYME MODELS BASED ON BORONIC ACIDS AND ON A MONOCLONAL ANTIBODY.
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Identifier
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AAI8801749
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identifier
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8801749
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Creator
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RAO, GALLA CHANDRASEKAR.
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Contributor
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Manfred Philipp
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Date
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1987
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Part IA. The hydrolysis of salicylidene-L-isoleucine (a Schiff's base) was accelerated in the presence of boronic acids. The pH dependence study shows that the boronic acids are catalysts when they are in their unionized acidic form. The catalytic rate constant is independent of pH, whereas binding depends on pH and decreases with increasing pH.;Hammett plots revealed that binding of boronic acids depends on sigma with a rho value of {dollar}-1.48.{dollar} Binding becomes better with electron-withdrawing substituents and is related to the pK of the boron acid. The catalytic rate constant is independent of substituent groups and rho is nearly zero ({dollar}-0.064{dollar}). The highest second-order rate constant obtained is 77 M{dollar}\sp{lcub}-1{rcub}{dollar} sec{dollar}\sp{lcub}-1{rcub}{dollar} and the lowest value is 0.09 M{dollar}\sp{lcub}-1{rcub}{dollar} sec{dollar}\sp{lcub}-1{rcub}{dollar}.;Part IB. Boronic acids also catalyze the hydrolysis of mandelonitrile. The rate of mandelonitrile hydrolysis increases with increasing pH. Boronic acids with electron-donating substituents catalyze the hydrolysis faster than with electron-withdrawing substituents. The Hammett rho is {dollar}-0.75.{dollar}.;Part II. TEPC-15 is a mouse myeloma protein that binds phosphorylcholine analogs. The phosphodiester group of phosphorylcholine is tetrahedral and resembles the transition state expected for the alkaline hydrolysis of esters. It was expected that TEPC-15 would hydrolyze choline esters by stabilizing the transition state of the hydrolysis. Several choline esters were used as potential substrates for the antibody. The carboxyl group of all these substrates is expected to bind in the site occupied by the phosphodiester group. The antibody did not accelerate the hydrolysis of any of those esters, but it binds them.;TEPC-15 did hydrolyze an ester-containing phosphorylcholine, the p-nitrophenyl ester of 6-(phosphorylcholine)hexanoic acid (PEPCH), where the ester linkage is expected to bind at a point distant from the phosphodiester binding site. The kinetic constants, K{dollar}\sb{lcub}\rm m{rcub}{dollar} and k{dollar}\sb{lcub}\rm max{rcub}{dollar}, obtained for the reaction of PEPCH with the antibody are 17 {dollar}\mu{dollar}M and 5.5 {dollar}\times{dollar} 10{dollar}\sp{lcub}-3{rcub}{dollar} sec{dollar}\sp{lcub}-1{rcub}{dollar}, respectively at pH 8.0. The rate of p-nitrophenolate ion release mediated by the antibody was pH-dependent and increases with increasing pH. The reaction was inhibited in the presence of phosphorylcholine analogs.;The antibody becomes inactive in the reaction with PEPCH and the inactive antibody was not reactivated even after treatment with hydroxylamine. These observations together with the pH profile of the reaction suggest that PEPCH acylates a lysine side chain near the antibody binding site.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biochemistry
