In:
Journal of the Chinese Chemical Society, Wiley, Vol. 51, No. 5B ( 2004-10), p. 1133-1140
Abstract:
Serine proteases are among the most studied enzymes for their role as model enzymes for studying the enzyme catalytic mechanism and medical interest in their inhibition. We have applied NMR methods to determine the structure, dynamics, and catalytic mechanism of a serine protease, E. coli thioesterase/protease I (TEP‐I). In this article we review the results of our efforts. We showed that TEP‐I is an / / type SNGH‐hydrolase with Ser 10 , Asp 154 and His 157 as the catalytic triad residues. In free TEP‐I, His 157 was found to forma strong hydrogen bond to Asp 154 , but not to Ser 10 ‐O H. Modelfree analysis of 15 N‐T 1 , 15 N‐T 2 and 1 H‐ 15 N NOE data revealed that TEP‐I is a rigid protein with a flexible catalytic binding pocket. Slow motion involving segments around the catalytic site was detected. The formation of Michaelis complex (MC) between TEP‐I and a transition state analogue, diethyl p‐nitrophenyl phosphate (DENP), and its subsequent conversion to the tetrahedral complex (TC) follow a two‐step process, a fast formation of MC followed by a slow conversion to TC. In both steps residues perturbed were confined mainly to four conserved segments comprising the active site. Comparable magnitudes of chemical shift perturbations were detected in both steps. From the large chemical shift perturbation upon conversion from MC to TC we proposed that the amide protons of Ser 10 and Gly 44 serve as the oxyanion‐hole proton donors to stabilize the tetrahedral adduct. The pattern of residues perturbed in both steps suggests a sequential, stepwise structural change upon binding of DENP.
Type of Medium:
Online Resource
ISSN:
0009-4536
,
2192-6549
DOI:
10.1002/jccs.200400168
Language:
English
Publisher:
Wiley
Publication Date:
2004
detail.hit.zdb_id:
2029538-8
