Interaction of nanoparticles with arginine kinase from Trypanosoma brucei: kinetic and mechanistic evaluation.
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 2.92 μmol min(-1) mg protein(-1), molecular mass of 40 kDa, temperature and pH optima of 30 °C and 7.8, and Km and Vmax as 2.94 mM and 0.161 μmol ml(-1) min(-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 1.5 nM (Ag) to 3.1 nM (Au). A mechanism for this inhibition was by interaction with Cys(271) positioned 3.3 Å from the reactive NH(1) 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.
Department of Biochemistry, Microbiology & Biotechnology Rhodes University, Grahamstown, South Africa.
Oluyomi Stephen
OS
Adeyemi
Chris George
CG
Whiteley
D001122:Arginine Kinase / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D015870:Gene Expression; D006046:Gold / Q000737:chemistry; D007700:Kinetics; D055595:Mechanical Phenomena; D053768:Metal Nanoparticles / Q000737:chemistry / Q000648:ultrastructure; D053758:Nanoparticles / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012834:Silver / Q000737:chemistry; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D011994:Recombinant Proteins; D012834:Silver; D006046:Gold; D001122:Arginine Kinase
Arginine kinase; Kinetic analysis; Mechanism; Silver, gold nanoparticles; Trypanosomiasis