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| - The Transition State of the Phosphoryl-Transfer Reaction Catalyzedby the Lambda Ser/Thr Protein Phosphatase
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| - The catalytic reaction of the Mn2+ form of the native bacteriophage λ phosphatase and the H76Nmutant was studied with the substrate p-nitrophenyl phosphate using heavy atom isotope effects and pH-dependent rate studies. The kinetic isotope effects in the substrate were measured at the nonbridging oxygenatoms [18(V/K)nonbridge], at the bridging oxygen atom undergoing bond cleavage [18(V/K)bridge], and at the nitrogenatom in the nitrophenol leaving group [15(V/K)]. The isotope effects with native enzyme at the pH optimumof 7.8 were 1.0133 ± 0.0006 for 18(V/K)bridge, 1.0006 ± 0.0003 for 15(V/K), and 0.9976 ± 0.0003 for18(V/K)nonbridge. These values were constant within experimental error across the pH range from 6.0 to 9.0 andwere also unchanged for the slower catalytic reaction resulting when Ca2+ was substituted for Mn2+. Theresults indicate that the chemical step of P−O bond cleavage is rate-limiting, the first metallophosphatase forwhich this has been shown to be the case. The isotope effects are very similar to those measured for reactionsof protein-tyrosine phosphatases, indicating that the two families of enzymes share similar dissociative transitionstates. The 18(V/K)bridge and 15(V/K) isotope effects for the H76N mutant were slightly increased in magnituderelative to the native enzyme but were much smaller than the values expected if the leaving group were departingwith a full negative charge. The pH vs kcat profile for the native enzyme is bell-shaped with pKa values of 7.7± 0.3 and 8.6 ± 0.4. Km values for substrate increased with pH approximately 70-fold across the pH range5.8−9.1. The Km for the H76N mutant was similar to that observed for native enzyme at high pH and wasrelatively constant across this pH range. The basic limb of the pH−rate profile is reduced but not abolishedin the H76N mutant reaction. The results are discussed in terms of the possible role of His-76 and the natureof the transition state for catalysis in the native enzyme and mutant.
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