The use of boronic acids in the study of hydrolytic enzymes.
Item
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Title
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The use of boronic acids in the study of hydrolytic enzymes.
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Identifier
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AAI9130308
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identifier
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9130308
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Creator
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de Soyza, Tushini Viveka.
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Contributor
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Adviser: Manfred Philipp
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Date
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1990
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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 I. Substrate specificity and boronic acid inhibition of E. coli RTEM-2 beta-lactamase. In this study, kinetic data for E. coli RTEM-2 beta-lactamase were obtained in order to understand the mechanism of action of this enzyme.;The substrate specificity of this Class A beta-lactamase for the two cephalosporins PADAC and nitrocefin was studied. This study revealed the presence of essential groups on the free enzyme which have pKs of 5.5 and 7.5. These pKs are tentatively assigned to the active site groups Glu-166 and Lys-73. pK{dollar}\sb2{dollar} was shifted by substrate binding, while pK{dollar}\sb1{dollar} was unchanged.;Inhibition of E. coli RTEM-2 beta-lactamase by boronic acids was rapid, occurring within a minute of mixing. Of the tested boronic acids, the arylboronic acids with bulky hydrophobic substituents were the most potent inhibitors. Of the other substituted phenylboronic acids, those with electron-withdrawing substituents were better inhibitors. The pH-dependence of inhibition for this group of compounds showed the ionization of a basic group with a pK of about 5.3. pK{dollar}\sb2{dollar} reflected the ionization of the substituted phenylboronic acid.;Part II. A comparison of the inhibition of the serine protease thrombin and the cysteine protease papain by a peptide boronic acid. Inhibition of bovine serum thrombin by 1-(Z-D-Phenylalanyl-L-Prolineamido)-1-dihydroxyborono-4-methoxybutane was competitive. Inhibition occurred soon after mixing, to an observation time of about a minute. The pH-dependence of inhibition was bell-shaped and was governed by a pK{dollar}\sb1{dollar} of 7.05, which is attributed to the ionization of the active site histidine. The pK{dollar}\sb2{dollar} of 8.75 reflects the ionization of the boronic acid group. The K{dollar}\sb{lcub}\rm i{rcub}{dollar} (Lim) was 6.9 nM.;Inhibition of bovine serum thrombin by Z-D-Phenylalanyl-L-Prolyl-L-Arginine was also competitive. The pH profile of inhibition was sigmoidal with a pK of 8.0. K{dollar}\sb{lcub}\rm i{rcub}{dollar} (Lim) was 1.5 {dollar}\mu{dollar}M. Inhibition was rapid.;Papain, a cysteine protease was also inhibited by the above peptide boronic acid. Inhibition occurred soon after mixing. However, inhibition was noncompetitive at pH 7. The pH profile of inhibition was bell-shaped and was governed by a pK{dollar}\sb1{dollar} of 4.4 and a pK{dollar}\sb2{dollar} of 7.4. K{dollar}\sb{lcub}\rm i{rcub}{dollar} (Lim) was 12.5 {dollar}\mu{dollar}M.
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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.
