Kinetic isotope effects for alkaline phosphatase reactions: implications for the role of active-site metal ions in catalysis

Abstract

Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active-site functional groups. In particular, the phosphate monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyzes phosphate monoester hydrolysis through a loose transition state, similar to that in solution. To gain further insight into the nature of the transition state and active-site interactions, we have determined kinetic isotope effects (KIEs) for AP-catalyzed hydrolysis reactions with several phosphate monoester substrates. The LFER and KIE data together provide a consistent picture for the nature of the transition state for AP-catalyzed phosphate monoester hydrolysis and support previous models suggesting that the enzymatic transition state is similar to that in solution. Moreover, the KIE data provides unique information regarding specific interactions between the transition state and the active-site Zn2+ ions. These results provide strong support for a model in which electrostatic interactions between the bimetallo Zn2+ site and a nonbridging phosphate ester oxygen atom make a significant contribution to the large rate enhancement observed for AP-catalyzed phosphate monoester hydrolysis.

Figure 1

Phosphate and phosphorothioate esters for which isotope effects are reported: red, nonbridge phosphoryl oxygen atoms [¹⁸(V/K)_nonbridge]; blue, bridge oxygen atom, site of bond fission [¹⁸(V/K)_bridge]; green, nitrogen atom of the leaving group [¹⁵(V/K)]. The structures are depicted with partial double bonds to emphasize that the nonbridging oxygen atoms that contribute to ¹⁸(V/K)_nonbridge are formally equivalent when the substrates are free in solution. For simplicity, the P-S and P-O bonds of pNPPS are depicted with the same partial double bond character, although it is likely that the P-S bond has more single bond character than the P-O bonds.^,

Figure 2

Compilation of kinetic data for the reactions of wt and R166S AP, represented by closed and open symbols, respectively. Reactions of alkyl phosphates (■, □) give values of β_lg of −0.85 ± 0.1 and −0.66 ± 0.1 respectively for reactions with wt and R166S AP.^, Reactions of aryl phosphates (♦) with wt AP are limited by a non-chemical step (dashed line). The rate constant for reaction of the aryl phosphate pNPP²⁻ (◊) with R166S AP deviates negatively from the value expected by extrapolation from the line for alkyl phosphates (dotted line and single-headed arrow). Rate constants for the reactions of mNBP²⁻ with wt and R166S AP (●, ○) deviate positively from the values expected from the line for alkyl phosphates (double-headed arrows).

Figure 3

KIEs for AP-catalyzed reactions are different from the corresponding solution reaction. A) Predicted direction of changes in KIEs relative to the solution reaction for two different models for behavior of phosphate monoester substrates in the AP active site. In model 1, the transition state is loose, similar to that in solution, and strong interactions with active site metal ions lead to decreases in both ¹⁸(V/K)_bridge and ¹⁸(V/K)_nonbridge. In model 2, AP catalyzes phosphate monoester hydrolysis through a tighter transition state than that in solution. This model predicts a decrease in ¹⁸(V/K)_bridge and an increase in ¹⁸(V/K)_nonbridge based on comparisons to corresponding solution reactions. B) The observed values of both ¹⁸(V/K)_bridge and ¹⁸(V/K)_nonbridge for AP-catalyzed pNPP²⁻ hydrolysis decrease relative to the corresponding solution reaction, consistent with model 2. Isotope effects are expressed as a percentage difference from unity [KIE_% = (KIE_obs −1)*100]. Values for the nonenzymatic reaction are shown in black and values for the AP-catalyzed reaction are shown in grey.

Scheme 1

Transition state model for phosphoryl transfer catalyzed by AP based on the structure with a bound vanadate ligand.^,

Scheme 2

Phosphate monoester hydrolysis through a loose transition state.

Scheme 3