Mutational and kinetic evaluation of conserved His-123 in dual specificityprotein-tyrosine phosphatase vaccinia H1-related phosphatase - Participation of Tyr-78 and Thr-73 residues in tuning the orientation of His-123

Active-site cysteine strategically positioned in the P-loop of protein-tyro sine phosphatases has been suggested to be further stabilized by hydrogen b onding arrays radiating out from the P-loop to neighboring residues. In thi s work, we investigated the structural, role of histidine array in HC(X)(5) RS motif of the (v) under bar accinia (H) under bar1-(r) under bar elated p rotein phosphatase (VHR), using site-directed mutagenesis in conjunction wi th an extensive kinetic analysis. Conserved His-123 was mutated along with neighboring residues Tyr-78 and Thr-73. The increased pK(alpha) values of a ctive-site Cys-124 found in Y78F and T73A mutants (6.51 and 6.75, respectiv ely) were comparable to those of H123A and H123F mutants. Kinetic evaluatio n of Y78F and T73A mutants further implicates that the mutations perturb th e relative position of Cys-124 within the P-loop. These results imply that Tyr-78 and Thr-73 make up an essential part of the Hi-123 array and structu rally tune the Cys-124 position. Tyr-78 of VHR turns out to be the invarian t Tyr reported in several protein-tyrosine phosphatases by a structure-base d sequence alignment, Therefore, orientation of the imidazole ring of His-1 23 by the invariant Tyr-78 is crucial for maintaining the proper position o f Cys-124 in the P-loop.