Mutation of Arg-166 of Alkaline Phosphatase Alters the Thio Effect but Not the Transition State for Phosphoryl Transfer. Implications for the Interpretation of Thio Effects in Reactions of Phosphatases

Abstract

It has been suggested that the mechanism of alkaline phosphatase (AP) is associative, or triester-like, because phosphorothioate monoesters are hydrolyzed by AP approximately 10(2)-fold slower than phosphate monoesters. This "thio effect" is similar to that observed for the nonenzymatic hydrolysis of phosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphate monoesters. The latter reactions proceed by loose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states. Wild-type alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate. In contrast, the R166A mutant alkaline phosphatase enzyme, in which the active site arginine at position 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster than p-nitrophenyl phosphorothioate. Despite this approximately 23-fold change in the magnitude of the thio effects, the magnitudes of Bronsted beta(lg) for the native AP (-0.77 +/- 0.09) and the R166A mutant (-0.78 +/- 0. 06) are the same. The identical values for the beta(lg) indicate that the transition states are similar for the reactions catalyzed by the wild-type and the R166A mutant enzymes. The fact that a significant change in the thio effect is not accompanied by a change in the beta(lg) indicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction. This result has important implications for the interpretation of thio effects in enzymatic reactions.

Kinetic Isotope Effects for Alkaline Phosphatase Reactions: Implications for the Role of Active-Site Metal Ions in Catalysis

“…11,20,67 Comparisons of phosphate and phosphorothioate monoesters have previously provided valuable insights into the AP reaction mechanism. 11,32,43 The KIEs for AP-catalyzed pNPPS 2− hydrolysis are significantly different from unity (Table 1), confirming previous conclusions that the chemical step is rate-determining for phosphorothioate hydrolysis catalyzed by both wt and R166S AP. 11,32 Furthermore, the KIEs for the wt and R166S AP-catalyzed reactions are identical within error.…”

“…10 Further, the values of β lg for reactions of aryl phosphorothioate monoesters with both wt and R166S AP are the same and similar to those for the alkyl phosphates. 11,32 These results are consistent with a similar loose transition state with substantial charge buildup on the leaving group oxygen for reactions of all phosphate monoester substrates, possibly with some lessening of the value of β lg relative to solution reactions due to the electrostatic interaction of the leaving group oxygen atom with an active site Zn 2+ ion (see also below). 12,13 The enzymatic reactivity of mNBP 2− can be directly compared with that of simple alkyl phosphates, as the leaving groups for these compounds fall in the same pK a range.…”

“…12 Because the leaving group oxygen for pNPP 2− is attached to a secondary carbon whereas the alkyl phosphate leaving groups are primary alkoxides, the deviation could arise from a steric effect. However, rate constants for substrates with p-nitrophenyl leaving groups also deviate negatively with respect to other aryl leaving groups in AP-catalyzed reactions of aryl phosphorothioates, 11,32 aryl sulfates, 13 methyl phenyl phosphate diesters, 44 and in several solution reactions. 44 This effect presumably arises from greater delocalization of the negative charge into the aromatic ring for p-nitrophenolate than for other aryl leaving groups.…”

Ground State Destabilization by Anionic Nucleophiles Contributes to the Activity of Phosphoryl Transfer Enzymes

“…Previous studies showed that mutation of Arg166, which interacts with two of the phosphoryl oxygen atoms (Figure 1), reduces P i binding affinity by ∼10 3 -fold at pH 8.0 ( = 460 µM and  = 640 µM, [35],[39];  = 0.5–1 µM, [16],[20],[38]). P i binding by the R166S AP single mutant could not be detected using the equilibrium-binding assay, as the concentrations of R166S AP needed to achieve binding in this assay are not readily obtained (Figure S7B).…”

QM/MM Analysis Suggests That Alkaline Phosphatase (AP) and Nucleotide Pyrophosphatase/Phosphodiesterase Slightly Tighten the Transition State for Phosphate Diester Hydrolysis Relative to Solution: Implication for Catalytic Promiscuity in the AP Superfamily

“…Experimentally, this mutant was used to avoid inhibition by P i , 22 and it is believed that the mutation does not alter the reaction mechanism of AP since LFERs are similar in the mutant and WT. 20,50 Moreover, the chemical step is fully rate-limiting in this mutant. We also study a double mutant, R166S/E322Y AP, which was constructed in recent experimental studies to analyze the contribution(s) from the Mg 2+ site in AP.…”