The catalytic reaction of the Mn2+ form of the native bacteriophage lambda phosphatase and the H76N mutant 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 oxygen atoms [(18)(V/K)(nonbridge)], at the bridging oxygen atom undergoing bond cleavage [(18)(V/K)(bridge)], and at the nitrogen atom in the nitrophenol 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), 1.0006 +/- 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 resulting when Ca2+ was substituted for Mn2+. The results indicate that the chemical step of P-O bond cleavage is rate-limiting, the first metallophosphatase for which this has been shown to be the case. The isotope effects are very similar to those measured for reactions of protein-tyrosine phosphatases, indicating that the two families of enzymes share similar dissociative transition states. The (18)(V/K)(bridge) and (15)(V/K) isotope effects for the H76N mutant were slightly increased in magnitude relative to the native enzyme but were much smaller than the values expected if the leaving group 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 across 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 this pH range. The basic limb of the pH-rate profile is reduced but not abolished in the H76N mutant reaction. The results are discussed in terms of the possible role of His-76 and the nature of the transition state for catalysis in the native enzyme and mutant.