The transition state of the phosphoryl-transfer reaction catalyzed by the lambda Ser/Thr protein phosphatase

The catalytic reaction of the Mn2+ form of the native bacteriophage lambda phosphatase and the H76N mutant was studied with the substrate p-nitropheny l phosphate using heavy atom isotope effects and pH-dependent rate studies. The kinetic isotope effects in the substrate were measured at the nonbridg ing oxygen atoms [(18)(V/K)(nonbridge)], at the bridging oxygen atom underg oing bond cleavage [(18)(V/K)(bridge)], and at the nitrogen atom in the nit rophenol leaving group [(15)(V/K)]. The isotope effects with native enzyme at the pH optimum of 7.8 were 1.0133 +/- 0.0006 for (18)(V/K)(bridge), 1.00 06 +/- 0.0003 for (15)(V/K), and 0.9976 +/- 0.0003 for (18)(V/K)(nonbridge) . These values were constant within experimental error across the pH range from 6.0 to 9.0 and were also unchanged for the slower catalytic reaction r esulting when Ca2+ was substituted for Mn2+. The results indicate that the chemical step of P-O bond cleavage is rate-limiting, the first metallophosp hatase for which this has been shown to be the case. The isotope effects ar e very similar to those measured for reactions of protein-tyrosine phosphat ases, indicating that the two families of enzymes share similar dissociativ e transition states. The (18)(V/K)(bridge) and (15)(V/K) isotope effects fo r the H76N mutant were slightly increased in magnitude relative to the nati ve enzyme but were much smaller than the values expected if the leaving gro up were departing with a full negative charge. The pH vs k(cat) profile for the native enzyme is bell-shaped with pK(a) values of 7.7 +/- 0.3 and 8.6 +/- 0.4. K-m values for substrate increased with pH approximately 70-fold a cross the pH range 5.8-9.1. The K-m for the H76N mutant was similar to that observed for native enzyme at high pH and was relatively constant across t his pH range. The basic limb of the pH-rate profile is reduced but not abol ished in the H76N mutant reaction. The results are discussed in terms of th e possible role of His-76 and the nature of the transition state for cataly sis in the native enzyme and mutant.