ROLES OF ASPARTIC-ACID-181 AND SERINE-222 IN INTERMEDIATE FORMATION AND HYDROLYSIS OF THE MAMMALIAN PROTEIN-TYROSINE-PHOSPHATASE PTP1

Protein tyrosine phosphatases (PTPases) share a number of conserved am ino acid residues, including the active site sequence HCXXGXXRS(T), wh ich are strongly implicated in catalysis. The roles of two conserved a ctive site residues, Asp-181 and Ser-222, were investigated using a co mbination of site-directed mutagenesis and kinetic analysis in the mam malian PTPase PTP1. The pH profiles for k(cat)/K-m and k(cat) of the w ild-type enzyme indicate that two ionizable groups, of pK(a) values 5. 1 and 5.44, must be deprotonated and one group with a pK(a) value of 4 .93 must be protonated for maximal activity. The group of pK(a) value 5.1 is the second ionization of the substrate phosphate moiety, Select ive thiolate anion inactivation indicates the residue with pK(a) value of 5.44 is C215. The pH-dependent profiles of the D181N mutant during establish the residue with pK(a) value of 4.93 to be Asp-181 and sugg est that it functions as a general acid phosphoryl transfer to the enz yme. Rapid reaction kinetics of wild-type and D181N mutant enzymes ind icate that the formation of the phospho-enzyme intermediate is rate-li miting at pH 7.0 and 30 degrees C. Enzymes containing the S222A mutati on exhibited rapid reaction burst kinetics, strongly suggesting that p hospho-enzyme intermediate hydrolysis is fully rate-limiting, The role of the active site S222 is to accelerate the rate of phospho-enzyme i ntermediate hydrolysis. The kinetic analysis of a third mutant, contai ning both the D181N and S222A mutations, suggests that D181 also serve s as a general base in the breakdown of the phospho-enzyme intermediat e.