| Abstract
| - Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceedthrough transition states that are altered from their solution counterparts, with the alterations presumablyarising from interactions with active-site functional groups. In particular, the phosphate monoester hydrolysisreaction 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 monoesterhydrolysis through a loose transition state, similar to that in solution. To gain further insight into the natureof the transition state and active-site interactions, we have determined kinetic isotope effects (KIEs) forAP-catalyzed hydrolysis reactions with several phosphate monoester substrates. The LFER and KIE datatogether provide a consistent picture for the nature of the transition state for AP-catalyzed phosphatemonoester hydrolysis and support previous models suggesting that the enzymatic transition state is similarto that in solution. Moreover, the KIE data provides unique information regarding specific interactions betweenthe transition state and the active-site Zn2+ ions. These results provide strong support for a model in whichelectrostatic interactions between the bimetallo Zn2+ site and a nonbridging phosphate ester oxygen atommake a significant contribution to the large rate enhancement observed for AP-catalyzed phosphatemonoester hydrolysis.
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