A substrate-assisted mechanism of nucleophile activation in a Ser-His-Asp containing C-C bond hydrolase.
The meta-cleavage product (MCP) hydrolases utilize a Ser-His-Asp triad to hydrolyze a carbon-carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ES(red), which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ES(red) decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2 (βnuc ∼ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His-Asp pair does not play an essential role. The data further suggest that ES(red) represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.
Departments of †Biochemistry & Molecular Biology and ‡Microbiology & Immunology, The University of British Columbia , Vancouver, British Columbia V6T 1Z4, Canada.
Antonio C
AC
Ruzzini
Shiva
S
Bhowmik
Subhangi
S
Ghosh
Katherine C
KC
Yam
Jeffrey T
JT
Bolin
Lindsay D
LD
Eltis
D000215:Acylation; D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D002853:Chromatography, Liquid; D004151:Dipeptides / Q000737:chemistry* / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000737:chemistry* / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D021241:Spectrometry, Mass, Electrospray Ionization; D020578:Sphingomonas / Q000201:enzymology*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004151:Dipeptides; D005231:Fatty Acids, Unsaturated; C422849:seryl-histidine; D001224:Aspartic Acid; C018971:2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid; D006867:Hydrolases