Sequential structural changes of Escherichia coli thioesterase/protease I in the serial formation of Michaelis and tetrahedral complexes with diethyl p-nitrophenyl phosphate

Sergiy I. Tyukhtenko, Alexandra V. Litvinchuk, Chi Fon Chang, Yu Chih Lo, Shin Jye Lee, Jei Fu Shaw, Yen Chywan Liaw, Tai Huang Huang

研究成果: Article同行評審

13 引文 斯高帕斯(Scopus)

摘要

Escherichia coli thioesterase/protease I (TEP-I) belongs to a new subclass of lipolytic enzymes of the serine hydrolase superfamily. Here we report the first direct NMR observation of the formation of the Michaelis complex (MC) between TEP-I and diethyl p-nitrophenyl phosphate (DENP), an active site directed inhibitor of serine protease, and its subsequent conversion to the tetrahedral complex (TC). NMR, ESI-MS, and kinetic data showed that DENP binds to TEP-I in a two-step process, a fast formation of MC followed by a slow conversion to TC. NMR chemical shift perturbation further revealed that perturbations were confined mainly to four conserved segments comprising the active site. Comparable magnitudes of chemical shift perturbations were detected in both steps. The largest chemical shift perturbation occurred around the catalytic Ser10 In MC, the conformation of the mobile Ser10 was stabilized, and its amide resonance became observable. From the large chemical shift perturbation upon conversion from MC to TC, we propose that the amide protons of Ser10 and Gly44 serve as the oxyanion hole proton donors that stabilize the tetrahedral adduct. The pattern of residues perturbed in both steps suggests a sequential, stepwise structural change upon binding of DENP. The present study also demonstrates the important catalytic roles of conserved residues in the SGNH family of proteins.

原文English
頁(從 - 到)8289-8297
頁數9
期刊Biochemistry
42
發行號27
DOIs
出版狀態Published - 2003 七月 15

All Science Journal Classification (ASJC) codes

  • 生物化學

指紋

深入研究「Sequential structural changes of Escherichia coli thioesterase/protease I in the serial formation of Michaelis and tetrahedral complexes with diethyl p-nitrophenyl phosphate」主題。共同形成了獨特的指紋。

引用此