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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...

The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865-2871]. The data reported here suggest that although Arg-166 is important for activity is not essential. The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme. First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished.

The proposed double in-line displacement mechanism of Escherichia coli alkaline phosphatase (AP) involving twometal ion catalysis is based on NMR spectroscopic and X-ray crystallographic studies. This mechanism is further supported by the X-ray crystal structures of the covalent phospho-enzyme intermediate of the H331Q mutant AP and of the transition state complex between the wild-type enzyme and vanadate, a transition state analog. Kinetic and structural studies on several genetically engineered versions of AP illustrate the overall importance of the active site's metal geometry, hydrogen bonding network and electrostatic potential in the catalytic mechanism.z 1999 Federation of European Biochemical Societies.

The active sites of aminopeptidase A (PepA) from Escherichia coli and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 Å and 1.6 Å resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions. It is shown that PepA is activated about 10-fold by bicarbonate when L-leucine p-nitroanilide is used as a substrate. No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases. A mutational analysis shows that Arg-356 inf luences activity by binding the bicarbonate ion but is not essential for activity. Mutation of the catalytic Lys-282 reduces k cat ͞K m about 10,000-fold.