Mutational and kinetic evaluation of conserved His-123 in dual specificity protein-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-tyrosine phosphatases has been suggested to be further stabilized by hydrogen bonding arrays radiating out from the P-loop to neighboring residues. In this work, we investigated the structural role of histidine array in HC(X)(5)RS motif of the vaccinia H1-related protein phosphatase (VHR), using site-directed mutagenesis in conjunction with an extensive kinetic analysis. Conserved His-123 was mutated along with neighboring residues Tyr-78 and Thr-73. The increased pK(a) values of active-site Cys-124 found in Y78F and T73A mutants (6.51 and 6.75, respectively) were comparable to those of H123A and H123F mutants. Kinetic evaluation of Y78F and T73A mutants further implicates that the mutations perturb the 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 His-123 array and structurally tune the Cys-124 position. Tyr-78 of VHR turns out to be the invariant Tyr reported in several protein-tyrosine phosphatases by a structure-based sequence alignment. Therefore, orientation of the imidazole ring of His-123 by the invariant Tyr-78 is crucial for maintaining the proper position of Cys-124 in the P-loop.
Kim
J H
JH
School of Chemistry and Molecular Engineering and Center for Molecular Catalysis, Seoul National University, Seoul 151-747, Korea.
Shin
D Y
DY
Han
M H
MH
Choi
M U
MU
D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D054637:Dual-Specificity Phosphatases; D005069:Evaluation Studies as Topic; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D017931:DNA Primers; D013912:Threonine; D014443:Tyrosine; D006639:Histidine; C517038:DUSP8 protein, human; D054637:Dual-Specificity Phosphatases; D017027:Protein Tyrosine Phosphatases