4.4 Article

A Substrate-Assisted Mechanism of Nucleophile Activation in a Ser-His-Asp Containing C-C Bond Hydrolase

期刊

BIOCHEMISTRY
卷 52, 期 42, 页码 7428-7438

出版社

AMER CHEMICAL SOC
DOI: 10.1021/bi401156a

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  1. Natural Sciences and Engineering Research Council (NSERC) of Canada
  2. NSERC PGS
  3. US DOE [W-31-109-Eng-38]
  4. NCRR [RR007707]
  5. NIGMS from the NIH [P41GM103543]

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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, ESred, 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 DxaB2-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 ESred 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 pK(a2) (beta(nuc) similar to 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 ESred 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.

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