title: Insights into the modulation of optimum pH by a single histidine residue in arginine deiminase from Pseudomonas aeruginosa.
abstract: Arginine deiminase (ADI) is a potential antitumor agent for the arginine deprivation treatment of L-arginine auxotrophic tumors. The optimum pH of ADI varies significantly, yet little is known about the origin of this variety. Here, Pseudomonas aeruginosa ADI (PaADI), an enzyme that functions only at acidic pH, was utilized as the model system. The results of UV-pH titration imply that the nucleophilic Cys406 thiol group is protonated in the resting state. The H405R single mutation resulted in an altered pH optimum (from pH 5.5 to 6.5), an increased k(cat) (from 9.8 s(-1) to 101.7 s(-1) at pH 6.5), and a shifted pH rate dependence (ascending limb pK(a) from 3.6 to 4.4). Other mutants were constructed to investigate the effects of hydrogen bonding, charge distribution, and hydrophobicity on the properties of the enzyme. The pH optima of His405 mutants were all shifted to a relatively neutral pH except for the H405E mutant. The results of kinetic characterizations and molecular dynamic simulations revealed that the active site hydrogen bonding network involving Asp280 and His405 plays an important role in controlling the dependence of PaADI activity on pH. Moreover, the H405R variant showed increased cytotoxicity towards arginine auxotrophic cancer cell lines.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Hanjing
authors.initials: H
authors.lastname: Ding
authors.forename: Hui
authors.initials: H
authors.lastname: Liu
authors.forename: Yan
authors.initials: Y
authors.lastname: Yin
authors.forename: Ying
authors.initials: Y
authors.lastname: Ding
authors.forename: Yan
authors.initials: Y
authors.lastname: Jia
authors.forename: Qingming
authors.initials: Q
authors.lastname: Chen
authors.forename: Guolin
authors.initials: G
authors.lastname: Zou
authors.forename: Zhongliang
authors.initials: Z
authors.lastname: Zheng
mesh_terms: D000595:Amino Acid Sequence; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D006639:Histidine; D006867:Hydrolases; C019723:arginine deiminase