Trapping the tetrahedral intermediate in the alkaline phosphatase reaction by substitution of the active site serine with threonine.

Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme that catalyzes the nonspecific hydrolysis of phosphomonoesters to an alcohol and inorganic phosphate (Pi). The enzyme is a dimer of two identical polypeptide chains, each of which contains 449 amino acids and two Zn2+ and one Mg2+ (Kim and Wyckoff 1989; Stec et al. 2000). The two zinc sites (Zn1 and Zn2) are 4 A apart, and the single magnesium site is located 5 A from Zn2 and 7 A from Zn1. In the structure of the wild-type noncovalent E•Pi complex (PDB code 1ALK; Kim and Wyckoff 1989), Pi is bound in the active site by interactions with the two zinc atoms and is also positioned in the active site through interactions with the guanidinium nitrogens of Arg166, the backbone nitrogen of Ser102, and a water-mediated interaction with Lys328. The catalytic mechanism for the alkaline phosphatase reaction was proposed in 1991 and revised in 2000 (Kim and Wyckoff 1991; Stec et al. 2000). The reaction proceeds through a series of four steps: The enzyme binds the substrate to form a noncovalent complex (E•ROP); the deprotonated Ser102 hydroxyl group attacks the phosphorus of the substrate to form a covalent serine–phosphate intermediate (E–Pi) (Schwartz and Lipmann 1961; Murphy et al. 1997); a hydroxide ion coordinated to Zn1 attacks the phosphorus to form a noncovalent enzyme–phosphate complex (E•Pi); and finally, Pi is released, reforming the free enzyme. The rate-determining step of the reaction is pH dependent; at acidic pH, the hydrolysis of the covalent E–P complex is rate-limiting, while under basic conditions the rate-limiting step becomes the dissociation of Pi from the E•Pi noncovalent complex (Hull et al. 1976; Gettins and Coleman 1983). In a variety of enzymes, Ser and Thr can often be interchanged for many catalytic functions, due to their structural similarity. Some hydrolytic enzymes, such as the asparaginases (Swain et al. 1993; Palm et al. 1996), the proteasome catalytic subunit (Lowe et al. 1995), and aspartyl glucosaminidase (Oinonen et al. 1995), use Thr as a nucleophile instead of Ser. These enzymes have a catalytic mechanism similar to that of the serine proteases, involving the formation of a covalent intermediate. The activation of a Thr nucleophile is different from a Ser nucleophile. Thr is typically activated by a primary amino group from the side chain of Lys or the N-terminal amino acid whereas Ser is activated as part of a catalytic triad. Although there are no significant chemical differences between the Oγ atoms of Thr and Ser, the side chains of these residues differ substantially in available conformations and size. The side chain of Thr may have an advantage in certain cases, in that the Cγ could help to exclude water from the active site (Dodson and Wlodawer 1998). Gijsbers et al. (2001) have also pointed out the structural and catalytic similarities between alkaline phosphatase and nucleotide pyrophosphatases/phosphodiesterases that utilize a Thr nucleotide in the reaction. Therefore, in this work we construct a mutant version of alkaline phosphatase that has the active site Ser replaced by Thr in order to investigate whether the enzyme can function with a Thr nucleophile. We combine functional characterization of the S102T alkaline phosphatase with two X-ray crystal structures (in the presence Pi and substrate) to understand how Thr functions as a catalytic group in the active site of alkaline phosphatase.