Analyzing the catalytic mechanism of protein tyrosine phosphatase PtpB from Staphylococcus aureus through site-directed mutagenesis.
Protein tyrosine phosphatase B (PtpB) from Staphylococcus aureus, MRSA 252, is a low molecular weight protein tyrosine phosphatase involved in its pathogenicity. PtpB has been modeled in silico and site-directed mutagenesis performed to ascertain the importance of active site residues Cys8, Arg14, Ser15 and Asp120 in its catalytic mechanism. Kinetic characterization of wild-type and the mutant PtpBs, C8S, R14A, S15T, S15A, D120A, D120E, D120N revealed the reaction mechanism followed by this LMWPTPase. The mutations caused major changes in the local environment resulting in significant decrease of its catalytic activity. Inhibition kinetics for the wild-type enzyme was performed with maleimide and maleimidobutyric acid.
Mukherjee
Somnath
S
Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India.
Dhar
Riddhiman
R
Das
Amit Kumar
AK
D000595:Amino Acid Sequence; D020148:Butyric Acid / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000201:enzymology*
D004791:Enzyme Inhibitors; D008301:Maleimides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D020148:Butyric Acid; C043592:maleimide; D017027:Protein Tyrosine Phosphatases; C547085:PtpB protein, S cerevisiae