ALTERING THE NUCLEOPHILE SPECIFICITY OF A PROTEIN-TYROSINE PHOSPHATASE-CATALYZED REACTION - PROBING THE FUNCTION OF THE INVARIANT GLUTAMINERESIDUES

Protein-tyrosine phosphatases (PTPases) catalysis involves a cysteinyl phosphate intermediate, in which the phosphoryl group cannot be trans ferred to nucleophiles other than water. The dual specificity phosphat ases and the low molecular weight phosphatases utilize the same chemic al mechanism far catalysis and contain the same (H/V)C(X)(5)R(S/T) sig nature motif present in PTPases, Interestingly, the latter two groups of phosphatases do catalyze phosphoryl transfers to alcohols in additi on to water, Unique to the PTPase family are two invariant Gin residue s which are located at the active site, Mutations at Gln-446 (and to a much smaller extent Gln-450) to Ala, Asn, or Met (but not Glu) residu es disrupt a bifurcated hydrogen bond between the side chain of Gln-44 6 and the nucleophilic water and confer phosphotransferase activity to the Yersinia PTPase, Thus, the conserved Gln-446 residue is responsib le for maintaining PTPases' strict hydrolytic activity and for prevent ing the PTPases from acting as kinases to phosphorylate undesirable su bstrates, This explains why phosphoryl transfer from the phosphoenzyme intermediate in PTPases can only occur to mater and not to other nucl eophilic accepters. Detailed kinetic analyses also suggest roles for G ln-446 and Gln-450 in PTPase catalysis. Although Gln-446 is not essent ial for the phosphoenzyme formation step, it plays an important role d uring the hydrolysis of the intermediate by sequestering and positioni ng the nucleophilic water in the active site for an in-line attack on the phosphorus atom of the cysteinyl phosphate intermediate. Gln-450 i nteracts through a bound water molecule with the phosphoryl moiety and may play a role for the precise alignment of active site residues, wh ich are important for substrate binding and transition state stabiliza tion for both of the chemical steps.