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Title: Substrate-Assisted Cysteine Deprotonation in the Mechanism of Dimethylargininase (DDAH) from Pseudomonas aeruginosa

Abstract

The enzyme dimethylargininase (also known as dimethylarginine dimethylaminohydrolase or DDAH; EC 3.5.3.18) catalyzes the hydrolysis of endogenous nitric oxide synthase inhibitors, N{sup {omega}}-methyl-L-arginine and N{sup {omega}},N{sup {omega}}-dimethyl-L-arginine. Understanding the mechanism and regulation of DDAH activity is important for developing ways to control nitric oxide production during angiogenesis and in many cases of vascular endothelial pathobiology. Several possible physiological regulation mechanisms of DDAH depend upon the presence of an active-site cysteine residue, Cys249 in Pseudomonas aeruginosa (Pa) DDAH, which is proposed to serve as a nucleophile in the catalytic mechanism. Through the use of pH-dependent ultraviolet and visible (UV-vis) difference spectroscopy and inactivation kinetics, the pK{sub a} of the active-site Cys249 in the resting enzyme was found to be unperturbed from pK{sub a} values of typical noncatalytic cysteine residues. In contrast, the pH dependence of k{sub cat} values indicates a much lower apparent pKa value. UV-vis difference spectroscopy between wild-type and C249S DDAH shows absorbance changes consistent with Cys249 deprotonation to the anionic thiolate upon binding positively charged ligands. The proton from Cys249 is lost either to the solvent or to an unidentified general base. A mutation of the active-site histidine residue, H162G, does not eliminate cysteine nucleophilicity, further arguing againstmore » a pre-formed ion pair with Cys249. Finally, UV-vis and X-ray absorption spectroscopy revealed that inhibitory metal ions can bind at these two active-site residues, Cys249 and His162, and also stabilize the anionic form of Cys249. These results support a proposed substrate-assisted mechanism for Pa DDAH in which ligand binding modulates the reactivity of the active-site cysteine.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
914102
Report Number(s):
BNL-78670-2007-JA
Journal ID: ISSN 0006-2960; BICHAW; TRN: US200804%%310
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 45; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CYSTEINE; NITRIC OXIDE; PSEUDOMONAS; ENZYME ACTIVITY; ENZYME INHIBITORS; HYDROLYSIS; BIOCHEMICAL REACTION KINETICS; national synchrotron light source

Citation Formats

Stone, E, Costello, A, Tierney, D, and Fast, W. Substrate-Assisted Cysteine Deprotonation in the Mechanism of Dimethylargininase (DDAH) from Pseudomonas aeruginosa. United States: N. p., 2006. Web. doi:10.1021/bi052595m.
Stone, E, Costello, A, Tierney, D, & Fast, W. Substrate-Assisted Cysteine Deprotonation in the Mechanism of Dimethylargininase (DDAH) from Pseudomonas aeruginosa. United States. https://doi.org/10.1021/bi052595m
Stone, E, Costello, A, Tierney, D, and Fast, W. 2006. "Substrate-Assisted Cysteine Deprotonation in the Mechanism of Dimethylargininase (DDAH) from Pseudomonas aeruginosa". United States. https://doi.org/10.1021/bi052595m.
@article{osti_914102,
title = {Substrate-Assisted Cysteine Deprotonation in the Mechanism of Dimethylargininase (DDAH) from Pseudomonas aeruginosa},
author = {Stone, E and Costello, A and Tierney, D and Fast, W},
abstractNote = {The enzyme dimethylargininase (also known as dimethylarginine dimethylaminohydrolase or DDAH; EC 3.5.3.18) catalyzes the hydrolysis of endogenous nitric oxide synthase inhibitors, N{sup {omega}}-methyl-L-arginine and N{sup {omega}},N{sup {omega}}-dimethyl-L-arginine. Understanding the mechanism and regulation of DDAH activity is important for developing ways to control nitric oxide production during angiogenesis and in many cases of vascular endothelial pathobiology. Several possible physiological regulation mechanisms of DDAH depend upon the presence of an active-site cysteine residue, Cys249 in Pseudomonas aeruginosa (Pa) DDAH, which is proposed to serve as a nucleophile in the catalytic mechanism. Through the use of pH-dependent ultraviolet and visible (UV-vis) difference spectroscopy and inactivation kinetics, the pK{sub a} of the active-site Cys249 in the resting enzyme was found to be unperturbed from pK{sub a} values of typical noncatalytic cysteine residues. In contrast, the pH dependence of k{sub cat} values indicates a much lower apparent pKa value. UV-vis difference spectroscopy between wild-type and C249S DDAH shows absorbance changes consistent with Cys249 deprotonation to the anionic thiolate upon binding positively charged ligands. The proton from Cys249 is lost either to the solvent or to an unidentified general base. A mutation of the active-site histidine residue, H162G, does not eliminate cysteine nucleophilicity, further arguing against a pre-formed ion pair with Cys249. Finally, UV-vis and X-ray absorption spectroscopy revealed that inhibitory metal ions can bind at these two active-site residues, Cys249 and His162, and also stabilize the anionic form of Cys249. These results support a proposed substrate-assisted mechanism for Pa DDAH in which ligand binding modulates the reactivity of the active-site cysteine.},
doi = {10.1021/bi052595m},
url = {https://www.osti.gov/biblio/914102}, journal = {Biochemistry},
issn = {0006-2960},
number = ,
volume = 45,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}