Spectroscopic and kinetic studies of the histidine residues of bovine low-molecular-weight phosphotyrosyl protein phosphatase.
The role of the two conserved histidine residues in the low-molecular-weight phosphotyrosyl protein phosphatases was investigated using site-directed mutagenesis of the recombinant bovine heart enzyme. His-66 and His-72 were individually mutated to alanine and to asparagine. A double mutant, containing only alanines in place of the histidines, was also created. The 1H NMR spectra of the purified proteins revealed no apparent tertiary structure alterations. Microscopic pKas for the two histidines were determined from a pH titration of the wild-type enzyme using 1H NMR spectroscopy and an MLEV-17 spectral editing scheme to more readily follow shifts in the specific histidine resonance peaks. His-66 titrates with an apparent pKa of 8.4 while for His-72 the value is 9.2. Since earlier chemical modification experiments indicated that the wild-type enzyme was inactivated by the histidine-selective modification reagent diethyl pyrocarbonate (DEP), the histidine mutants were tested for sensitivity to DEP. Both of the histidine single mutants were inactivated by DEP, and surprisingly, the double mutant containing no histidines was also readily inactivated by DEP. Thus, for this protein, modification by DEP is not specific for histidine residues. Kinetic studies of the mutant proteins reveal that neither histidine is essential in the catalytic mechanism. His-66 mutants showed virtually identical catalytic properties compared to wild-type enzyme, whereas His-72 mutants had reduced specific activity and higher phosphate Ki and lower Km values at pH5 and higher. It is proposed that His-72, although not essential for catalysis, may serve a significant structural role at the enzyme active site.
Davis
J P
JP
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393.
Zhou
M M
MM
Van Etten
R L
RL
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers; D004047:Diethyl Pyrocarbonate; D006639:Histidine / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D006639:Histidine; D017027:Protein Tyrosine Phosphatases; D004047:Diethyl Pyrocarbonate