1999 Fiscal Year Final Research Report Summary
Generation of functional molecules which regulate the glutathione level in living organisms-Design and synthesis of specific inhibitors of γ-glutamylcysteine synthetase-
| Project/Area Number |
10680566
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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 |
Bioorganic chemistry
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
HIRATAKE Jun Institute for Chemical Research, Kyoto University, Assoc. Prof., 化学研究所, 助教授 (80199075)
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| Project Period (FY) |
1998 – 1999
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| Keywords | glutathione biosynthesis / γ-glutamylcysteine synthetase / transition-state analogue inhibitor / γ-glutamylphosphate intermediate analogue / mechanism-based enzyme inactivator / enzyme suicide substrate / γ-glutamyltranspeptidase |
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| Research Abstract |
The purpose of this research is to design and synthesis of functional molecules which affects the glutathione level in living organisms. The biosynthesis and degradation of glutathione are mediated by γ-glutamylcysteine synthetase (γ-GCS) and γ-glutamyltranspeptidase (GGT), respectively. Based on the reaction mechanisms of these enzymes, specific inhibitors of these two enzymes were designed, synthesized and evaluated in terms of inhibition potency and profile. γ-GCS catalyzes the reaction by activating the γ-carboxyl group of L-Glu by phosphorylation, followed by nucleophilic substitution with L-Cys. As a transition-state analogue of the substitution step, tetrahedral phosphinic acid and sulfoximine analogues were synthesized. Each compound was found to be a potent time-and ATP-dependent inactivator of γ-GCS. In particular, the sulfoximine served as an extremely powerful slow-binding inhibitor, in which the sulfoximine S=NH nitrogen was phosphorylated by ATP in a mechanism-based manne
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r to form a phosphorylated sulfoximine which is highly analogous to the putative transition state. Two diastereomeric sulfoximines with respect to the chiral sulfur atom were synthesized and evaluated as an inhibitor of γ-GCS. The diastereomer with (R)-sulfur atom served as a highly potent ATP-dependent inactivator (KィイD2iィエD2=39 nM), but the (S)-diastereomer was a rather weak reversible inhibitor (KィイD2iィエD2=12μM). Thus, the enzyme recognized the chirality of the sulfur atom and phosphorylated the (R)-sulfoximine solely. The (R)-sulfoximine inhibited γGCS 1000 times more strongly than buthionine sulfoximine, a famous inhibitor of γ-GCS. In an attempt to obtain an enzyme-suicidal substrate of γ-GCS, the effect of L-glutamic acid γ-hydroxamate was examined. This compound was found to inhibit γ-GCS very slowly, but irreversibly in the presence of ATP, and the inhibition profile suggested that the hydroxamate was phosphorylated enzymatically by ATP to form an isocyanate via a Rossen-type rearrangement. A transition-state analogue inhibitor of GGT was also synthesized. A γ-phosphonofluoridate analogue of Glu was synthesized and was found to serve as a mechanism-based labeling agent of GGT to phosphonylate the active site catalytic nucleophile. Ion-spray MS of the labeled GGT revealed that the N-terminal Thr-391 in the small subunit was the catalytic nucleophile of GGT. Less
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Research Products
(12 results)
