Mutational study of human phosphohistidine phosphatase: effect on enzymatic activity.
Although protein histidine phosphorylation is estimated to account for about 6% of total protein phosphorylation in eukaryotes, knowledge on histidine phosphorylation and dephosphorylation is still limited. Recently, a few reports have appeared on a mammalian 14-kDa phosphohistidine phosphatase, also named protein histidine phosphatase. Molecular cloning of the protein has opened possibilities for exploring its properties and physiological role. In the present work, we have searched for potential active site residues in the human phosphohistidine phosphatase by point mutations of conserved histidine and arginine residues to alanine. When assayed by the phosphohistidine-containing peptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide, mutants H53A and H102A showed no detectable activity. Compared to the wild-type recombinant enzyme, the specific activity of mutant R45A was decreased by one order of magnitude, that of mutant R78A was decreased by about 30%, while that of mutant H81A was essentially unchanged. These results will facilitate future studies of the reaction mechanism, substrate binding, and molecular structure of the phosphohistidine phosphatase.
Ma
Ruixin
R
Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden.
Kanders
Erik
E
Sundh
Ulla Beckman
UB
Geng
Meiyu
M
Ek
Pia
P
Zetterqvist
Orjan
O
Li
Jin-Ping
JP
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D004789:Enzyme Activation; D019143:Evolution, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases