A conserved hydrogen-bond network in the catalytic centre of animal glutaminyl cyclases is critical for catalysis

Kai Fa Huang, Yu Ruei Wang, En Cheng Chang, Tsung Lin Chou, Andrew H.J. Wang

研究成果: 雜誌貢獻文章同行評審

24 引文 斯高帕斯(Scopus)

摘要

QCs (glutaminyl cyclases; glutaminyl-peptide cyclotransferases, EC 2.3.2.5) catalyse N-terminal pyroglutamate formation in numerous bioactive peptides and proteins. The enzymes were reported to be involved in several pathological conditions such as amyloidotic disease, osteoporosis, rheumatoid arthritis and melanoma. The crystal structure of human QC revealed an unusual H-bond (hydrogen-bond) network in the active site, formed by several highly conserved residues (Ser160, Glu201, Asp248, Asp 305 and His319), within which Glu201 and Asp248 were found to bind to substrate. In the present study we combined steady-state enzyme kinetic and X-ray structural analyses of 11 single-mutation human QCs to investigate the roles of the H-bond network in catalysis. Our results showed that disrupting one or both of the central H-bonds, i.e., Glu201 ⋯Asp305 and Asp248 ⋯Asp305, reduced the steady-state catalysis dramatically. The roles of these two COOH⋯COOH bonds on catalysis could be partly replaced by COOH⋯water bonds, but not by COOH⋯CONH2 bonds, reminiscent of the low-barrier Asp⋯Asp H-bond in the active site of pepsin-like aspartic peptidases. Mutations on Asp305, a residue located at the centre of the H-bond network, raised the Km value of the enzyme by 4.4-19-fold, but decreased the kcat value by 79-2842-fold, indicating that Asp305 primarily plays a catalytic role. In addition, results from mutational studies on Ser160 and His319 suggest that these two residues might help to stabilize the conformations of Asp248 and Asp305 respectively. These data allow us to propose an essential proton transfer between Glu201, Asp305 and Asp248 during the catalysis by animal QCs.

原文英語
頁(從 - 到)181-190
頁數10
期刊Biochemical Journal
411
發行號1
DOIs
出版狀態已發佈 - 4月 1 2008
對外發佈

ASJC Scopus subject areas

  • 生物化學
  • 分子生物學
  • 細胞生物學

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