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Analyzing the catalytic mechanism of MPtpA: A low molecular weight protein tyrosine phosphatase from Mycobacterium tuberculosis through site‐directed mutagenesis
Author(s) -
Madhurantakam Chaithanya,
Chavali Venkata Ramana Murthy,
Das Amit Kumar
Publication year - 2007
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21816
Subject(s) - sodium orthovanadate , sodium molybdate , protein tyrosine phosphatase , mycobacterium tuberculosis , site directed mutagenesis , biochemistry , mutant , mutagenesis , chemistry , tyrosine , active site , enzyme , sodium tungstate , virulence , phosphatase , biology , molybdate , tuberculosis , gene , medicine , organic chemistry , pathology , tungsten
Mycobacterium tuberculosis adopts various measures to escape from the hostile environment of the host cells. A low molecular weight protein tyrosine phosphatase (LMWPTPase) MPtpA was found to be active in virulent mycobacterial forms during the phagocytosis process. To ascertain the importance of conserved residues Cys11, Arg17, and Asp126 in the catalytic mechanism of MPtpA, site‐directed mutagenesis was performed, namely C11S, R17A, D126A, and D126N. Kinetic characterization of wild‐type and the mutant MPtpAs using para ‐nitrophenyl phosphate revealed the reaction mechanism followed by this LMWPTPase and it is similar to the other PTPases. All the LMWPTPases have a common signature motif, ‘C(X) 5 R(S/T)’ and an Asp as the general acid residue and the mechanism followed by MPtpA can be aptly attributed to other LMWPTPases as well, considering the similar three‐dimensional conformation. We have shown that the mutations caused major changes in the chemical environment surrounding the mutated residues and resulted in the decrease of catalytic activity significantly. Inhibition kinetics was performed with phosphate analogues: sodium molybdate, sodium orthovanadate, and sodium tungstate. Proteins 2008. © 2007 Wiley‐Liss, Inc.