Chemistry and Chemical Biology ETDs

Publication Date

1-20-1978

Abstract

The role of the metal ion in rat erythrocyte glyoxalase I was evaluated. The apoenzyme was formed by dialysis against an EDTA-imidazole buffer. Fluorescence studies of the Mg++- and Mn++-dansylated glyoxalase I's suggest that the metal ion is near the active site of the enzyme and participates in the catalytic reaction. Stereochemical studies of the conversion of methylglyoxal to S-lactoylglutathione with Mg++, Mn++, Co++ and Ni++-glyoxalase I's show that the 0-lactate of S-lactoylglutathione (97±1.5%) is formed. This result suggests that the reaction mechanism is the same with a change in metal ion. Human erythrocyte samples were screened on polyacrylamide­starch slab gel electrophoresis using a glyoxalase I specific dye to determine if the sample contained a slow-migrating (AA) or a fast migrating (BB) glyoxalase I or all three glyoxalase I isozymes (AA, AB and BB). The glyoxalase I's obtained from two homozygotes are more labile than the combination of the three glyoxalase I isozymes obtained from the heterozygote. All glyoxalase I samples were purified at least 10,000 fold, and each of the isozymes has a molecular weight of 44,000. Human erythrocyte glyoxalase I's from a heterozygote show broad substrate specificity. Isotope studies with glyoxalase I's from a heterozygote show that removal of the α-hydrogen atom of the hemimercaptal is the rate-determining step of the reaction. Comparative kinetic studies show the glyoxalase I isozymes from the two homozygotes and the combination of glyoxalase I isozymes obtained from the heterozygote to be kinetically indistinguishable. Human erythrocyte glyoxalase I and Hela cell glyoxalase I are competitively inhibited by MS-3,3',4’-dihydroxymethyl-5’-hydroxy-6’-(3-methyl-2-butenyl)-phenyl-2,4-dihydroxy-6-methylbenzoate. The combination of glyoxalase I isozymes from the heterozygote has a KI with MS-3 which is 2-3 fold greater than the KI for the glyoxalase I's from the two homozygotes. HeLa cell glyoxalase I, which is electrophoretically similar to the BB glyoxalase I, has a KI (3.4 ± 0.3 x 10-6 M) which is similar to the KI of the glyoxalase I's from the two homozygotes. HeLa cell glyoxalase I also has the same Km using methylglyoxal as substrate as human erythrocyte glyoxalase I.

Language

English

Document Type

Dissertation

Degree Name

Chemistry

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

David Lee Vander Jagt

Second Committee Member

William Fletcher Coleman

Third Committee Member

Beulah Marie Woodfin

Fourth Committee Member

Philip Reyes

Fifth Committee Member

Jimmy Clayton Standefer

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