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Purification and properties of gamma-glutamyltranspeptidase from Proteus mirabilis
Abstract:
gamma-Glutamyltranspeptidase was purified ca. 15,200-fold from cell-free extracts of Proteus mirabilis to electrophoretic homogeneity and then crystallized. The enzyme has an estimated molecular weight of 80,000 and consists of two different subunits with molecular weights of ca. 47,000 and 28,000. The purified enzyme catalyzed hydrolysis and transpeptidation of various gamma-glutamyl compounds, including the oxidized and reduced forms of glutathione, gamma-glutamyl compounds of L-phenylalanine, L-tyrosine, L-histidine, L-alpha-aminobutyrate, L-leucine, and p-nitroaniline. Glycylglycine, L-phenylalanine, L-methionine, L-histidine, L-tryptophan, and L-isoleucine were good acceptors of the gamma-glutamyl moiety in the transpeptidation reaction. Km values for gamma-glutamyl compounds were on the order of 10(-4) to 10(-5) M, and those for acceptor peptides and amino acids were on the order of 10(-2) to 10(-3) M. The enzyme was inhibited by L-serine plus borate and 6-diazo-5-oxo-L-norleucine, which are inhibitors of gamma-glutamyltranspeptidases isolated from mammals. Various amino acids alone were found to inhibit the transpeptidation competitively with a gamma-glutamyl donor. Kinetic analysis suggested that the reaction sequence of substrate binding and product release proceeds according to a ping pong bi bi mechanism.
Insights
This study purified gamma-glutamyltranspeptidase from Proteus mirabilis, revealing its subunit structure and enzymatic activity. The enzyme
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Gamma-glutamyltranspeptidase (GGT) plays a crucial role in amino acid metabolism and transport.
- Understanding GGT from microbial sources can offer insights into enzyme evolution and function.
- Proteus mirabilis is a common bacterium, and its GGT characterization is of interest.
Purpose of the Study:
- To purify and characterize gamma-glutamyltranspeptidase from Proteus mirabilis.
- To determine the enzyme's kinetic properties and substrate specificity.
- To compare microbial GGT with mammalian counterparts.
Main Methods:
- Purification of gamma-glutamyltranspeptidase to electrophoretic homogeneity.
- Crystallization of the purified enzyme.
- Enzyme activity assays for hydrolysis and transpeptidation.
- Determination of kinetic parameters (Km) and inhibition studies.
- Subunit molecular weight determination via SDS-PAGE.
Main Results:
- Gamma-glutamyltranspeptidase was purified ~15,200-fold and crystallized.
- The enzyme has a molecular weight of 80,000 Da, composed of 47,000 Da and 28,000 Da subunits.
- Catalyzed hydrolysis and transpeptidation of various gamma-glutamyl compounds.
- Demonstrated substrate acceptance by various amino acids and peptides.
- Enzyme kinetics followed a ping pong bi bi mechanism.
- Inhibited by known mammalian GGT inhibitors (L-serine plus borate, 6-diazo-5-oxo-L-norleucine).
Conclusions:
- Proteus mirabilis gamma-glutamyltranspeptidase shares functional and inhibitory similarities with mammalian enzymes.
- The characterized enzyme exhibits broad substrate specificity for gamma-glutamyl compounds and amino acid acceptors.
- The ping pong bi bi mechanism provides a framework for understanding the enzyme's catalytic cycle.