A catalytic mechanism for cysteine N-terminal nucleophile hydrolases, as revealed by free energy simulations.

Alessio Lodola,Marco Mor,Luigi Capoferri,Daniele Piomelli,Marco De Vivo,Andrea Cavalli,Davide Branduardi,Matthew H Todd

doi:10.1371/journal.pone.0032397

Abstract

The N-terminal nucleophile (Ntn) hydrolases are a superfamily of enzymes specialized in the hydrolytic cleavage of amide bonds. Even though several members of this family are emerging as innovative drug targets for cancer, inflammation, and pain, the processes through which they catalyze amide hydrolysis remains poorly understood. In particular, the catalytic reactions of cysteine Ntn-hydrolases have never been investigated from a mechanistic point of view. In the present study, we used free energy simulations in the quantum mechanics/molecular mechanics framework to determine the reaction mechanism of amide hydrolysis catalyzed by the prototypical cysteine Ntn-hydrolase, conjugated bile acid hydrolase (CBAH). The computational analyses, which were confirmed in water and using different CBAH mutants, revealed the existence of a chair-like transition state, which might be one of the specific features of the catalytic cycle of Ntn-hydrolases. Our results offer new insights on Ntn-mediated hydrolysis and suggest possible strategies for the creation of therapeutically useful inhibitors.

Highlights

N-terminal nucleophile (Ntn-) hydrolases are a superfamily of enzymes specialized in the cleavage of amide bonds [1]
Ntnhydrolases become catalytically active after autocatalytic cleavage of an N-terminal peptide, which creates a novel N-terminal residue – usually a Ser, Thr, or Cys – that is responsible for amide bond cleavage [2]
To elucidate the catalytic mechanism of this class of enzymes, we investigated the first step of taurodeoxycholate (TAU) hydrolysis by conjugated bile acid hydrolase (CBAH) (Figure 1B) using a hybrid quantum mechanics/molecular mechanics (QM/MM) technique [20,21], a well-established approach in computational enzymology [22,23]

Summary

Introduction

N-terminal nucleophile (Ntn-) hydrolases are a superfamily of enzymes specialized in the cleavage of amide bonds [1]. The reaction starts with a proton transfer between the nucleophile (OH or SH) of the catalytic residue and the alpha-amino group of the same amino acid (Figure 1A).

Results

Conclusion