A Model of the Transition State in the Alkaline Phosphatase Reaction

Abstract

A high resolution crystal structure of Escherichia coli alkaline phosphatase in the presence of vanadate has been refined to 1.9 Å resolution. The vanadate ion takes on a trigonal bipyramidal geometry and is covalently bound by the active site serine nucleophile. A coordinated water molecule occupies the axial position opposite the serine nucleophile, whereas the equatorial oxygen atoms of the vanadate ion are stabilized by interactions with both Arg-166 and the zinc metal ions of the active site. This structural complex supports the in-line displacement mechanism of phosphomonoester hydrolysis by alkaline phosphatase and provides a model for the proposed transition state in the enzymecatalyzed reaction. Escherichia coli alkaline phosphatase (AP)1 is a homodimeric metalloenzyme catalyzing the nonspecific hydrolysis of phosphate monoesters into inorganic phosphate and an alcohol. The overall structure is approximately 100 Å ϫ 50 Å ϫ 50 Å with the active sites 30 Å apart from each other on opposite sides of the molecule. Each active site contains two tightly bound zinc ions (Zn 1 and Zn 2 ) and one magnesium ion. The three closely spaced metal binding sites trace a triangle with the two zinc ions 4 Å apart and the magnesium binding site approximately 5 Å from Zn 2 and 7 Å from Zn 1 . The active site pocket shown in Fig. 1 (top panel) with bound inorganic phosphate is shallow and, in addition to the three metal ions, is lined by Arg-166 and Ser-102.Based on the structure of the enzyme with phosphate bound, Kim and Wyckoff (1) have proposed the reaction mechanism shown in Scheme 1. The free enzyme interacts with a phosphomonoester (ROP) to form the Michaelis enzyme-substrate complex (E⅐ROP). This complex breaks down upon nucleophilic attack of Ser-102 on the phosphate group of the substrate forming the covalent phospho-enzyme intermediate (E-P). This covalent intermediate is subsequently hydrolyzed into the noncovalent enzyme-phosphate complex (E⅐P i ). As predicted by the mechanism, the reaction proceeds with overall retention of configuration at the phosphorus center. The two consecutive in-line displacement steps are postulated to have trigonal bipyramidal transition states.In this study the interaction between vanadate (i.e. orthovanadate, VO 4 3Ϫ ) and E. coli alkaline phosphatase (EC 3.1.3.1) is evaluated by x-ray crystallography. Vanadate, an oxyanion of pentavalent vanadium, readily adopts a five-coordinate geometry resembling the proposed transition state in the enzyme-catalyzed reaction of alkaline phosphatase. In fact, vanadate has been used as a transition state analog in several enzyme-catalyzed reactions including phosphomonoester hydrolysis by rat acid phosphatase and ribonucleotide phosphodiester hydrolysis by ribonuclease A (2, 3). Vanadate is isostructural and isoelectronic with phosphate, a product, and a competitive inhibitor of alkaline phosphatase (4). Unlike the phosphate ion, vanadate can form stable five-coordinate species (4) allowing it to serve as a transition state analog for both the ac...

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

“…The results obtained for the red kidney bean enzyme resemble those obtained for the di-Zn II alkaline phosphatase from E. coli, where vanadate acts as a competitive inhibitor with K ic = 12 μmol L -1 at pH 8.0. 25 The crystal structure of this enzyme, complexed with vanadate, has been solved and supports the hypothesis that vanadate is a suitable mimic of the proposed fivecoordinate phosphoryl transition state (Figure 1). Based on the similarity of the inhibition data it appears likely that red kidney bean PAP has a transition state similar to that observed in E. coli alkaline phosphatase.…”

“…23 The di-zinc alkaline phosphatase from Escherichia coli is inhibited by vanadate with similar efficiency (K i = 12 μmol L -1 at pH 8.0), and the crystal structure of the enzyme-inhibitor complex shows that vanadate binds in a penta-coordinate geometry to the active site with three of its oxygen atoms coordinating the two metal ions in the active site in a tripodal arrangement (Figure 1). 25 In this work, the inhibitory effects of fluoride and vanadate on the activity of red kidney bean and pig PAP are assessed and compared with those measured for other PAPs. The mechanistic implications of these studies are also discussed.…”

“…21 For metallohydrolases, fluoride and vanadate are known inhibitors which have been shown to coordinate directly to the metal ion(s) in the active site and thus interfere with catalytic turnover. 1,20,[23][24][25][26][27][28] The two inhibitors are likely to affect different aspects of catalysis and hence may provide insight into different steps of the reaction mechanism.…”

“…21 For metallohydrolases, fluoride and vanadate are known inhibitors which have been shown to coordinate directly to the metal ion(s) in the active site and thus interfere with catalytic turnover. 1,20,[23][24][25][26][27][28] The two inhibitors are likely to affect different aspects of catalysis and hence may provide insight into different steps of the reaction mechanism.The ability of fluoride to readily replace nucleophilic hydroxide groups can provide (i) an insight into the number of labile water/hydroxide binding sites in the active site, and (ii) the role of the terminally bound or bridging hydroxides in the reaction mechanism. 26,29 Fluoride, a well established osteogenic agent which promotes osteoblastic proliferation and differentiation at clinically relevant concentrations, inhibits animal PAPs in a pH-dependent manner.…”

Cellular function and molecular structure of ecto-nucleotidases

“…The first transition state is stabilized by the two zinc ions and R166. A crystallographic model for the transition state has been obtained in the form of a co-crystal structure of EcAP with vanadate [598]. The vanadate is covalently bound to the serine nucleophile and adopts a trigonal bipyramidal geometry.…”