Metal Specificity Is Correlated with Two Crucial Active Site Residues in Escherichia coli Alkaline Phosphatase †,‡ Jie Wang, Kimberly A. Stieglitz, and Evan R. Kantrowitz* Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467 ReceiVed January 26, 2005; ReVised Manuscript ReceiVed March 18, 2005 ABSTRACT: Escherichia coli alkaline phosphatase exhibits maximal activity when Zn 2+ fills the M1 and M2 metal sites and Mg 2+ fills the M3 metal site. When other metals replace the zinc and magnesium, the catalytic efficiency is reduced by more than 5000-fold. Alkaline phosphatases from organisms such as Thermotoga maritima and Bacillus subtilis require cobalt for maximal activity and function poorly with zinc and magnesium. Previous studies have shown that the D153H alkaline phosphatase exhibited very little activity in the presence of cobalt, while the K328W and especially the D153H/K328W mutant enzymes can use cobalt for catalysis. To understand the structural basis for the altered metal specificity and the ability of the D153H/K328W enzyme to utilize cobalt for catalysis, we determined the structures of the inactive wild-type E. coli enzyme with cobalt (WT_Co) and the structure of the active D153H/K328W enzyme with cobalt (HW_Co). The structural data reveal differences in the metal coordination and in the strength of the interaction with the product phosphate (P i ). Since release of P i is the slow step in the mechanism at alkaline pH, the enhanced binding of P i in the WT_Co structure explains the observed decrease in activity, while the weakened binding of P i in the HW_Co structure explains the observed increase in activity. These alterations in P i affinity are directly related to alterations in the coordination of the metals in the active site of the enzyme. Alkaline phosphatase (EC 3.1.3.1) is a nonspecific phos- phomonoesterase found in organisms from all kingdoms of life. The mechanism of the alkaline phosphatase reaction involves the attack of a serine alkoxide on the phosphorus of the substrate to form a transient covalent enzyme- phosphate complex followed by the hydrolysis of the serine phosphate (1). Alignment of the sequences from a selection of alkaline phosphatases shows that the enzymes are very well conserved, especially near the active site. Upon comparison of residues within 10 Å of the phosphate position in the active site of the Escherichia coli enzyme to the corresponding residues in other alkaline phosphatases, the majority of enzymes are found to conserve most of these amino acid positions. The residues that directly interact with the substrate, Ser102 and Arg166, are conserved in all cases. In the E. coli enzyme, the active site contains two Zn 2+ ions and one Mg 2+ ion. The residues interacting with the zinc at the M1 1 site (Asp327, His331, and His412) and the residues interacting with zinc at the M2 site (Asp51, Asp369, and His370) are conserved in all compared sequences. The only variations occur at amino acids Asp153 and Lys328 near the E. coli enzyme Mg 2+ binding site (M3). Invariably, the only change observed at E. coli position 153 is from an Asp to a His. The most common change at E. coli alkaline phosphatase position 328 is to a Trp, the exception being a His found in the Pyrococcus abyssi and eukaryotic enzymes. In addition to alkaline phosphatases that utilize Zn 2+ and Mg 2+ , there are some that utilize cobalt (2, 3). Among the Co 2+ -requiring enzymes, Thermotoga maritima alkaline phosphatase and the Bacillus subtilis phoAIII and phoAIV gene products have His and Trp at E. coli positions 153 and 328, respectively. To test whether positions 153 and 328 alone or in combination determine the metal specificity of alkaline phosphatase, we previously prepared by site-specific mutagenesis the D153H and K328W mutations in the E. coli enzyme individually and in combination (4). The wild-type and D153H enzymes showed very little activity in the presence of Co 2+ . For example, when the Zn 2+ and Mg 2+ are replaced with cobalt in the wild-type enzyme, the k cat decreases more than 500-fold (Table 1). However, the k cat of the cobalt-containing K328W enzyme is only ∼5- † This work was supported in part by Grant GM42833 from the National Institutes of Health. Data for this study were measured at beamlines X12b and X26c of the National Synchrotron Light Source. Financial support comes principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the U.S. Department of Energy, and from the National Center for Research Resources of the National Institutes of Health. ‡ Coordinates and observed structure factor amplitudes for the structures described in this paper have been deposited in the Protein Data Bank (entries 1Y6V and 1Y7A). * To whom correspondence should be addressed. E-mail: evan.kantrowitz@bc.edu. Telephone: (617) 552-4558. Fax: (617) 552- 2705. 1 Abbreviations: M1, highest-affinity metal site occupied by Zn 2+ in the wild-type enzyme; M2, second metal site occupied by Zn 2+ in the wild-type enzyme; M3, lowest-affinity metal site occupied by Mg 2+ in the wild-type enzyme; WT_Zn/Mg, wild-type E. coli alkaline phosphatase with Zn 2+ in the M1 and M2 sites and Mg 2+ in the M3 site (PDB entry 1ED8); WT_Co, wild-type E. coli alkaline phosphatase with Co 2+ in the M1-M3 sites (PDB entry 1Y6V); HW_Co, mutant version of E. coli alkaline phosphatase with Asp153 replaced with His and Lys328 replaced with Trp with Co 2+ in the M1-M3 sites (PDB entry 1Y7A); HH_Zn, mutant version of E. coli alkaline phosphatase with Asp153 and Lys328 are replaced with His with Zn 2+ in the M1- M3 sites (PDB entry 1ANI). 8378 Biochemistry 2005, 44, 8378-8386 10.1021/bi050155p CCC: $30.25 © 2005 American Chemical Society Published on Web 05/18/2005