Probing the Enzyme Catalytic Mechanism by Nuclear Magnetic Resonance ‐ A Case Study of a Serine Protease

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 a /b /a -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.