Structure and Mechanism of Alkaline Phosphatase

Abstract

Alkaline phosphatase was the first zinc enzyme to be discovered in which three closely spaced metal ions (two Zn ions and one Mg ion) are present at the active center. Zn ions at all three sites also produce a maximally active enzyme. These metal ions have center-to-center distances of 3.9 A (Zn1-Zn2), 4.9 A (Zn2-Mg3), and 7.1 A (Zn1-Mg3). Despite the close packing of these metal centers, only one bridging ligand, the carboxyl of Asp51, bridges Zn2 and Mg3. A crystal structure at 2.0-A resolution of the noncovalent phosphate complex, E.P, formed with the active center shows that two phosphate oxygens form a phosphate bridge between Zn1 and Zn2, while the two other phosphate oxygens form hydrogen bonds with the guanidium group of Arg166. This places Ser102, the residue known to be phosphorylated during phosphate hydrolysis, in the required apical position to initiate a nucleophilic attack on the phosphorous. Extrapolation of the E.P structure to the enzyme-substrate complex, E.ROPO4(2-), leads to the conclusion that Zn1 must coordinate the ester oxygen, thus activating the leaving group in the phosphorylation of Ser102. Likewise, Zn2 appears to coordinate the ester oxygen of the seryl phosphate and activate the leaving group during the hydrolysis of the phosphoseryl intermediate. Both of these findings suggest that there may be a significant dissociative character to each of the two displacements at phosphorous catalyzed by alkaline phosphatase. A water molecule (or hydroxide) coordinated to Zn1 following formation of the phosphoseryl intermediate appears to be the nucleophile in the second step of the mechanism. Dissociation of the product phosphate from the E.P intermediate is the slowest, 35 s-1, and therefore the rate-limiting, step of the mechanism at alkaline pH. Since the determination of the initial crystal structure of alkaline phosphatase, two other crystal structures of enzymes involved in phosphate ester hydrolysis have been completed that show a triad of closely spaced zinc ions present at their active centers. These enzymes are phospholipase C from Bacillus cereus (structure at 1.5-A resolution) (43) and P1 nuclease from Penicillium citrinum (structure at 2.8-A resolution) (74). Both enzymes hydrolyze phosphodiesters. Substrates for phospholipase C are phosphatidylinositol and phosphatidylcholine, while P1 nuclease is an endonuclease hydrolyzing single stranded ribo- and deoxyribonucleotides. P1 nuclease also has activity as a phosphomonoesterase against 3'-terminal phosphates of nucleotides. The Zn ions in both enzymes form almost identical trinuclear sites.(ABSTRACT TRUNCATED AT 400 WORDS)

A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase

“…Interestingly, the pH optimum for the Pt oxidation reaction is substantially lower (pH 7) than it is for phosphate ester hydrolysis (pH Ͼ 8.5) (ref. 27 and K.Y. and W.W.M, unpublished data).…”

“…This observation includes the so-called ''metal-free'' H 2 -forming methylene tetrahydromethanopterin dehydrogenase of methanogenic archaea, which is now known to contain Fe as well (41). In contrast, BAP contains no redox active metals, although both Zn and Mg are required for hydrolytic activity (27). Treatment of BAP with chelators inhibits Pt oxidation, suggesting that metals play a role in the Pt reaction (data not shown).…”

“…The E. coli phoA gene encodes the secreted, periplasmic enzyme BAP. BAP is an exhaustively studied, nonspecific phosphomonoesterase that allows E. coli to use phosphate esters as alternative sources of P (27,28). Consistent with the known enzymatic function of BAP, our original hypothesis regarding the phoA-dependent pathway was that Pt would be oxidized to a phosphate ester by as-yet-unknown enzymes and then hydrolyzed by BAP to release the free phosphate needed for growth.…”

“…Although small amounts of phosphate were produced by the eukaryotic phosphatases after overnight incubation, only E. coli BAP was able to catalyze the reaction at significant rates. This finding is particularly surprising because the eukaryotic enzymes are actually much better phosphatases, with specific activities up to 40-fold higher than BAP (27). These enzymes all share significant homology (25-35% identity) with the E. coli BAP; furthermore, most of the active-site residues, including Ser-102 (which forms a covalent phospho-enzyme intermediate during the phosphatase reaction), are conserved (27).…”

Genetic Complexity, Structure, and Characterization of Highly Active Bovine Intestinal Alkaline Phosphatases

“…Cloning of AP cDNAs from a variety of species and comparison of their primary structures has revealed a high degree of sequence conservation and even a 25-30% similarity between E. coli and mammalian APs (2,3). The three-dimensional crystallographic structure of the E. coli AP homodimer has been solved (4,5) and the reaction mechanism inferred (6). Efforts to crystallize any of the mammalian APs have so far been unsuccessful.…”