The pH-dependent activation mechanism of Ser102 in Escherichia coli alkaline phosphatase: a theoretical study.

Abstract

The accepted catalytic mechanism of alkaline phosphatases was established on the hypothesis that Mg-coordinated water is a readily available hydroxide ion that functions as a general base and accepts a proton from the Ser102 hydroxyl group. The roles of the two distinctive residues Asp153 and Lys328 in Escherichia coli alkaline phosphatase (ECAP), which distinguish it from the mammalian enzymes, are elusive. Based on the crystal structures of ECAP in the absence and presence of inorganic phosphate, we have investigated the activation of Ser102 for nucleophilic attack and the subsequent formation of a covalent phosphoseryl intermediate using hybrid density functional theory (DFT) with the B3LYP functional. Our calculations confirmed a proton transfer path from the hydroxyl group of Ser102 to the carboxyl group of Asp153 via water-mediated interactions through which Ser102 can be activated for nucleophilic attack. Our calculations further suggest that the activation mechanism of Ser102 is pH dependent, which explains why ECAP achieves its maximum activity at an alkaline pH for enzyme-catalysed reactions. This activation mechanism can also be extended to all APs with similar active-site structures. Our study, for the first time, provides the definite activation mechanism of Ser102 in ECAP, which is universal for all APs and explains the alkaline phosphatase activity of these enzymes. This work also sheds new light on the relationship between enzyme-catalytic mechanisms and enzyme-catalytic properties.