| Co-Investigator(Kenkyū-buntansha) |
KABASHIMA Tsutomu Nagasaki University, Graduate School of Biomedical Sciences, Assistant, 大学院・医歯薬学総合研究科, 助手 (20274673)
ITO Kiyoshi Nagasaki University, Graduate School of Biomedical Sciences, Associate Professor, 大学院・医歯薬学総合研究科, 助教授 (50201926)
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| Budget Amount *help |
¥13,900,000 (Direct Cost: ¥13,900,000)
Fiscal Year 2002: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2001: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2000: ¥6,800,000 (Direct Cost: ¥6,800,000)
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| Research Abstract |
In order to clarify the mechanism of substrate specificity of proline specific peptidase, prolyl aminopeptidase was chosen as a target. Molecular cloning of the gene and crystal structure of the prolyl aminopeptidase (EC 3.4:11.5) from Serratia marcescens has been studied by us (1. Biochem.122, 601-605 (1997), ibid 126, 559-565 (1999)). Through these studies, Phe139, Tyr149, Glu204, and Arg136 were estimated to concern with substrate recognition. To elucidate the details of the mechanism for substrate specificity, site-directed mutagenesis method was applied. F139A mutant showed 80-fold decreased in catalytic efficiency (kcat/Km), but Y149A mutant did not make significant changes of catalytic efficiency. The catalytic efficiency of E204Q mutant was about 4% of the value of wild type. Peptidase activity of the mutant (R136A) was decreased markedly, however, arylamidase activity using Pyr-bNA was retained as the wild-enzyme. By these results, it was clarified that the pyrrolidine ring an
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d the amino group of proline in S1 site were recognized by Phe139 and Glu204, respectively. P1' of substrate was recognized by Arg136. On the other hand, the enzyme had two cysteine residues. Mutants C74A and C271A were inhibited by PCMB, but double mutated enzyme (C74/271A) was inert against it.
On the other hand, mechanism of pyroglutamyl peptidase was studied. In order to clarify the mechanism of substrate recognition for the unique structure of the pynolidone ring, X-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl peptidase bound to a transition-state analog inhibitor, pyroglutaminal, was determined. Two hydrogen bounds were located between the main chain of the enzyme and the inhibitor and the pynolidone ring of the inhibitor was inserted into the hydrophobic pocket. These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bounds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is more important to the hydrophobic interaction than Phe-142 and furthermore Phe-13 serves as a "induced-fit".
Thus suggested similar mechanism of substrate recognition for five members ring was estimated. Less
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