Metal Specificity Is Correlated with Two Crucial Active Site Residues in Escherichia coli Alkaline Phosphatase

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• 作者：Jie Wang ; Kimberly A. Stieglitz ; and Evan R. Kantrowitz
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：June 14, 2005
• 年：2005
• 卷：44
• 期：23
• 页码：8378 - 8386
• 全文大小：614K
• 年卷期：v.44,no.23(June 14, 2005)
• ISSN：1520-4995

文摘

Escherichia coli alkaline phosphatase exhibits maximal activity when Zn²⁺ fills the M1 andM2 metal sites and Mg²⁺ fills the M3 metal site. When other metals replace the zinc and magnesium, thecatalytic efficiency is reduced by more than 5000-fold. Alkaline phosphatases from organisms such asThermotoga maritima and Bacillus subtilis require cobalt for maximal activity and function poorly withzinc and magnesium. Previous studies have shown that the D153H alkaline phosphatase exhibited verylittle activity in the presence of cobalt, while the K328W and especially the D153H/K328W mutant enzymescan use cobalt for catalysis. To understand the structural basis for the altered metal specificity and theability of the D153H/K328W enzyme to utilize cobalt for catalysis, we determined the structures of theinactive wild-type E. coli enzyme with cobalt (WT_Co) and the structure of the active D153H/K328Wenzyme with cobalt (HW_Co). The structural data reveal differences in the metal coordination and in thestrength of the interaction with the product phosphate (P_i). Since release of P_i is the slow step in themechanism at alkaline pH, the enhanced binding of P_i in the WT_Co structure explains the observeddecrease in activity, while the weakened binding of P_i in the HW_Co structure explains the observedincrease in activity. These alterations in P_i affinity are directly related to alterations in the coordinationof the metals in the active site of the enzyme.