Insights into the modulation of optimum pH by a single histidine residue in arginine deiminase from Pseudomonas aeruginosa
-
Hanjing Ding
and
Zhongliang Zheng
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 kcat (from 9.8 s-1 to 101.7 s-1 at pH 6.5), and a shifted pH rate dependence (ascending limb pKa 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.
©2012 by Walter de Gruyter Berlin Boston
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Articles in the same Issue
- Masthead
- Masthead
- Guest Editorial
- Highlight: The universe of proteolytic networks and mechanisms
- Highlight: 7th General Meeting of the International Proteolysis Society
- Current and prospective applications of non-proteinogenic amino acids in profiling of proteases substrate specificity
- Understanding the substrate specificity of conventional calpains
- Protease-dependent mechanisms of complement evasion by bacterial pathogens
- An ensemble view of thrombin allostery
- Processive proteolysis by γ-secretase and the mechanism of Alzheimer’s disease
- TMPRSS4 is a type II transmembrane serine protease involved in cancer and viral infections
- A catalogue of putative HIV-1 protease host cell substrates
- Identifi cation of protease exosite-interacting peptides that enhance substrate cleavage kinetics
- A plant Kunitz-type inhibitor mimics bradykinin-induced cytosolic calcium increase and intestinal smooth muscle contraction
- Characterisation and metabolism of astroglia-rich primary cultures from cathepsin K-deficient mice
- Disruption of gingipain oligomerization into non-covalent cell-surface attached complexes
- Review
- Synthesis and biological actions of diphosphoinositol phosphates (inositol pyrophosphates), regulators of cell homeostasis
- Minireviews
- Redox Biology on the rise
- Adipose triglyceride lipase in immune response, inflammation, and atherosclerosis
- Research Articles/Short Communications
- Protein Structure and Function
- Insights into the modulation of optimum pH by a single histidine residue in arginine deiminase from Pseudomonas aeruginosa
