The catalytic role of aspartic acid-92 in a human dual-specific protein-tyrosine-phosphatase.
The mechanism of catalysis for the human dual-specific (vaccinia H1-related) protein-tyrosine-phosphatase was investigated. The pH dependence of the kcat value is bell-shaped when p-nitrophenyl phosphate was employed as a model substrate. The kcat/Km pH profile rises with a slope of 2 and decreases with a slope of -1, indicating that two groups must be unprotonated and one group must be protonated for activity. An amino acid residue with an apparent pKa value of 5.5 +/- 0.2 must be unprotonated and a residue with a pKa value of 5.7 must be unprotonated for activity. The pKa value of the catalytic cysteine-124 (C124) was 5.6 +/- 0.1. The aspartic acid-92-asparagine (D92N) mutant enzyme was 100-fold less active than the native enzyme and exhibited the loss of the basic limb in the pH profiles, suggesting that in the native enzyme D92 must be protonated for activity. The D92 residue is conserved throughout the entire family of dual-specific phosphatases. Mutants glutamic acid-6-glutamine, glutamic acid-32-glutamine, aspartic acid-14-asparagine, and aspartic acid-110-asparagine had less than a 2-fold effect on the kinetic parameters when compared to native enzyme. Based upon the lack of a "burst" in rapid reaction kinetics, formation of the intermediate is rate-limiting with both native and D92N mutant enzymes. In agreement with rate-limiting formation of the intermediate, the pKa value of 5.5 for the group which must be unprotonated for activity was assigned to C124.(ABSTRACT TRUNCATED AT 250 WORDS)
Denu
J M
JM
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Zhou
G
G
Guo
Y
Y
Dixon
J E
JE
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics; D054640:Dual Specificity Phosphatase 3; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009596:Nitrophenols / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D007461:Iodoacetates; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D001224:Aspartic Acid; C008644:nitrophenylphosphate; D004247:DNA; C517025:DUSP3 protein, human; D054640:Dual Specificity Phosphatase 3; D017027:Protein Tyrosine Phosphatases; D019807:Iodoacetic Acid