Substrate entering and product leaving trajectories predict an engulfing dynamic for the major conformational change of the &amp;946;-lactam acylase

Xi Huang,Wei-Hong Jiang,Guo-Ping Zhao,Jun Yin,Zixin Deng

doi:10.1038/npre.2008.2514.1

Abstract

AbstractIt is still a major challenge to acquire insight into the conformational changes between the ground state and the transition state of an enzyme, although conformational fluctuation within interconverting conformers has been widely investigated (1-4). Here, we utilize different enzymatic reactions in b-lactam acylase to figure out the substrate/product trajectories in the enzyme, thereby probing the overall conformational changes in transition state. First, an auto-proteolytic intermediate of cephalosporin acylase (EC 3.5.1.11) with partial spacer segment was identified. As a final proteolytic step, the deletion of this spacer segment was revealed to be a first-order reaction, suggesting an intramolecular Ntn mechanism for the auto-proteolysis. Accordingly, the different proteolytic sites in the acylase precursor indicate a substrate entering pathway along the spacer peptide. Second, bromoacyl-7ACA can interact with penicillin G acylase (EC 3.5.1.11) in two distinguish aspects, to be hydrolyzed as a substrate analogue and to affinity alkylate the conserved Trpb4 as a product analogue. The kinetic correlation between these two reactions suggests a channel opening from Serb1 to Trpb4, responsible for the main product leaving. These two reaction trajectories relaying at the active centre, together with the crystal structures (5-10), predict an engulfing dynamic involving pocket constriction and channel opening.

Highlights

It is still a major challenge to acquire insight into the conformational changes between the ground state and the transition state of an enzyme, conformational fluctuation within interconverting conformers has been widely investigated 1-4
It is unlikely that the following step(s) for the free spacer deletion adopt either the proposed intermolecular N-terminal nucleophile (Ntn) mechanism 16,17 or the intramolecular interaction with Gluα[161] as nucleophile that was speculated from the spacer peptide with 9 amino acid residues 21, because cephalosporin acylase (CA) has no effects on the removal of the free spacer peptide of its mutant 21 and the length of CA spacer is still uncertain 13,16,21
The smaller one is a normal α-subunit (CAα), whose sequence is TPQ...RTLGE as determined by the MALDI-TOF spectra (Supplementary Fig. 1) and the MS/MS spectrum of its C-terminal trypic fragment TLGE (Supplementary Fig. 2). This confirms that the spacer peptide in this study is consisted of 10 amino-acid residues, GDPPDLADQG 13

Summary

Introduction

It is still a major challenge to acquire insight into the conformational changes between the ground state and the transition state of an enzyme, conformational fluctuation within interconverting conformers has been widely investigated 1-4. Besides the normal substrate hydrolysis, the auto-proteolysis[13] of the spacer peptide in the pocket and the affinity alkylation 14 on the Trpβ[4] in the tightly packed αββα motif (Fig. 1) would indicate the substrate entering and product leaving pathway in the enzyme.

Results

Conclusion