Department of Biochemistry, Microbiology and Biotechnology Rhodes University, Grahamstown, South Africa
Adeyemi, O.S., Department of Biochemistry, Microbiology and Biotechnology Rhodes University, Grahamstown, South Africa; Whiteley, C.G., Department of Biochemistry, Microbiology and Biotechnology Rhodes University, Grahamstown, South Africa
Arginine kinase is not only absent from mammalian hosts but is critical to the survival of trypanosomes under stressful conditions and consequently its inhibition may lead to an effective treatment for trypanosomiasis. The His-tagged enzyme was cloned from Trypanosoma brucei genomic DNA, expressed in Escherichia coli BL21 DE3 cells and purified on a Ni-affinity column and by FPLC on a Superdex 200 HR. The enzyme had a specific activity of 2.92μmolmin-1mgprotein-1, molecular mass of 40kDa, temperature and pH optima of 30°C and 7.8, and Km and Vmax as 2.94mM and 0.161μmolml-1min-1 (arginine substrate). The interaction of the enzyme with silver and gold nanoparticles showed a non-competitive inhibition with, respectively, 75% and 62% decrease in activity; Ki values ranged from 1.5nM (Ag) to 3.1nM (Au). A mechanism for this inhibition was by interaction with Cys271 positioned 3.3Å from the reactive NH1 of substrate arginine. This cysteine controls electrophilic and nucleophilic character of the guanidinium group that is crucial for enzymatic phosphoryl transfer between ADP and ATP. © 2013 Elsevier B.V.
adenosine diphosphate; adenosine triphosphate; arginine; arginine kinase; cysteine; genomic DNA; gold nanoparticle; guanidine; histidine; nickel; silver nanoparticle; article; competitive inhibition; controlled study; electrophilicity; enzyme activity; enzyme analysis; enzyme inhibitor interaction; enzyme kinetics; enzyme substrate complex; Escherichia coli; fast protein liquid chromatography; molecular cloning; molecular weight; nonhuman; nucleophilicity; pH; temperature; Trypanosoma brucei; trypanosomiasis; Arginine kinase; Kinetic analysis; Mechanism; Silver, gold nanoparticles; Trypanosomiasis; Arginine Kinase; Cloning, Molecular; Enzyme Activation; Gene Expression; Gold; Kinetics; Mechanical Phenomena; Metal Nanoparticles; Nanoparticles; Protein Binding; Recombinant Proteins; Silver; Trypanosoma brucei brucei