Probing the Origin of the Compromised Catalysis of E. coliAlkaline Phosphatase in its Promiscuous Sulfatase Reaction

Authors

Irina Catrina
Patrick J. O'Brien
Jamie Purcell
Ivana Nikolic-Hughes
Jesse G. Zalatan
Alvan C. Hengge, Utah State UniversityFollow
Daniel Herschlag

Document Type

Article

Journal/Book Title

Journal of the American Chemical Society

Publication Date

2007

Volume

129

Issue

17

First Page

5760

Last Page

5765

Abstract

The catalytic promiscuity of E. coli alkaline phosphatase (AP) and many other enzymes provides a unique opportunity to dissect the origin of enzymatic rate enhancements via a comparative approach. Here, we use kinetic isotope effects (KIEs) to explore the origin of the 10⁹-fold greater catalytic proficiency by AP for phosphate monoester hydrolysis relative to sulfate monoester hydrolysis. The primary ¹⁸O KIEs for the leaving group oxygen atoms in the AP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) and p-nitrophenylsulfate (pNPS) decrease relative to the values observed for nonenzymatic hydrolysis reactions. Prior linear free energy relationship results suggest that the transition states for AP-catalyzed reactions of phosphate and sulfate esters are “loose” and indistinguishable from that in solution, suggesting that the decreased primary KIEs do not reflect a change in the nature of the transition state but rather a strong interaction of the leaving group oxygen atom with an active site Zn²⁺ ion. Furthermore, the primary KIEs for the two reactions are identical within error, suggesting that the differential catalysis of these reactions cannot be attributed to differential stabilization of the leaving group. In contrast, AP perturbs the KIE for the nonbridging oxygen atoms in the reaction of pNPP but not pNPS, suggesting a differential interaction with the transferred group in the transition state. These and prior results are consistent with a strong electrostatic interaction between the active site bimetallo Zn²⁺ cluster and one of the nonbridging oxygen atoms on the transferred group. We suggest that the lower charge density of this oxygen atom on a transferred sulfuryl group accounts for a large fraction of the decreased stabilization of the transition state for its reaction relative to phosphoryl transfer.

Recommended Citation

Irina Catrina, Patrick J. O’Brien, Jamie Purcell, Ivana Nikolic-Hughes, Jesse G. Zalatan, Alvan C. Hengge and Daniel Herschlag. “Probing the Origin of the Compromised Catalysis of E. coli Alkaline Phosphatase in its Promiscuous Sulfatase Reaction.” J. Am. Chem. Soc. 2007; 129(17); 5760-5765.