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 k(cat) value is bell-shaped when p-nitrophenyl phosphate was employed as a model substrate. The k(cat)/K-m 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 pK(a) value of 5.5 +/- 0.2 must be unprotonated and a residue with a pK(a) value of 5.7 must be unprotonated for activity. The pK(a) 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 pK(a) value of 5.5 for the group which must be unprotonated for activity was assigned to C124. The D92 residue acts as a general acid by protonating the phenolate ion in the rate-limiting formation of the intermediate. D92 may also serve as general base by abstracting a proton from a water molecule in the hydrolysis of the phosphoenzyme intermediate.