Structural study of active center of γ-glutamyltranspeptidase and its reaction mechanism.
| Project/Area Number |
15580061
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied microbiology
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| Research Institution | Kyoto University |
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Principal Investigator |
SUZUKI Hideyuki Kyoto University, Graduate School of Biostudies, Associate Professor, 生命科学研究科, 助教授 (10202136)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | glutathione / γ-glutamyltranspeptidase / active center / Ntn-hydrolase / nucleophile / substrate specificity |
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| Research Abstract |
γ-Glutamyltranspeptidase(GGT) utilizes both D and L-γ-glutamyl compounds as substrates. Kinetic parameters were measured using D/L-glutamine and D/L-γ-glutamyl-p-nitroanilide. Km values against D compounds were about 10 times bigger that that against L-compounds. This indicates that there are residues recognizing both amino group and carbonyl group on α carbon. The amino acid sequences of GGTs and class IV cephalosporin acylases have high similarity. Especially, residues conserved among GGTs are well conserved in class IV cephalosporin acylases. Among them, Asp-433, whose corresponding residue inhuman GGT was suggested to interact with α-amino group of γ-glutamyl compounds, was substituted with Asn since this residue is Asn in class IV cephalosporin acylases. The mutant GGT obtained GL-7-ACA acylase activity and we succeeded in converting GGT to class IV cephalosporin acylase. When the corresponding residue in Bacillus subtilis GGT was substituted with Ala, the mutant GGT lost the transpeptidation activity while hydrolysis activity was remained. Using GGT crystals, we had modeled 90% residues of E.coli GGT.
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Report
(3 results)
Research Products
(29 results)
