Catalytic mechanism of Escherichia coli alkaline phosphatase: resolution of three variants of the acyl-enzyme mechanism

Abstract

Three variants of the classical acyl-enzyme mechanism were compared theoretically with respect to the predicted transient kinetics of substrate hydrolysis by Escherichia coli alkaline phosphatase. In all three, acyl-enzyme hydrolysis was assumed to exist initially primarily as a noncovalent complex with the acid product, inorganic phosphate. In one mechanism, the pre-steady-state rate-controlling step was assumed to be the dissociation of acid product from its initial complex with enzyme. In the other two, pre-steady-state rate control was assigned to an enzyme isomerization occurring before or after substrate binding to free enzyme. Under concentration conditions of excess substrate and acid product, integrated rate laws were used to reject the possibility of pre-steady-state rate control by enzyme isomerization between phosphate dissociation and substrate binding. Whereas this mechanism predicts a pre-steady-state noncompetitive relationship between substrate and acid product, the stopped-flow kinetics of 4-methylumbelliferyl phosphate hydrolysis demonstrates a competitive relationship, consistent with either of the other two mechanisms. Under concentration conditions of stoichiometrically limiting substrate, computer simulations eliminated the possibility of rate control by enzyme isomerization after substrate binding. This mechanism predicts a substrate concentration dependence for the apparent first-order rate constant of substrate hydrolysis which disagrees with previously published data [Halford, S. E. (1971) Biochem. J. 125, 319--327]; the other two mechanisms are consistent with experiment. Comparison of transient kinetic theory and experiment under these two contrasting concentration conditions suggests strongly that the rate-controlling step in phosphate ester hydrolysis by E. coli alkaline phosphate is the dissociation of "sticky" acid product from its noncovalent complex with enzyme. This mechanism explains an anomaly in the stopped-flow kinetic trace, a substoichiometric pre-steady-state burst of alcohol product release.

Quantum-Mechanical Study on the Catalytic Mechanism of Alkaline Phosphatases

“…On the other hand, for alkyl phosphate substrates the computed values point to the nucleophilic attack of the serine alkoxide to phosphorus ( k 2 ) as the rate-determining step. While for aryl phosphates the rate-determining step corresponds to product release ( k 4 in Scheme ) at pH > 7.5 and to hydrolysis of the phosphoserine ( k 3 ) at pH < 7.5, − phosphorylation of the enzyme ( k 2 ) has been hinted as rate-limiting for alkyl phosphates . Hence, the present computational results are in accordance with the mechanistic interpretations arising from experimental observations.…”

“…In regard to the second chemical step ( k 3 ), the presence of a more basic alkyl oxide anion would help the proton transfer from the Zn 1 -coordinated water molecule to generate the nucleophilic hydroxide (step 3 in Scheme ), assisting the hydrolysis of the phosphoserine intermediate; on the other hand, formation of the hydroxide ion would be less facile in the presence of an aryl substrate (step 3 in Scheme ). Therefore, the second energy barrier becomes the highest for aryl phosphates, with the enzymatic rate-determining step being highly influenced by the pH of the medium (at pH < 7.5, k 3 is rate-limiting; at pH > 7.5, k 4 is rate-limiting). − A p K a of 8.0 has been suggested for the zinc-coordinated water …”

“…The rate-limiting step is pH-dependent for aryl phosphate substrates, as the hydrolysis of the covalent intermediate E–P ( k 3 , Scheme ) is rate-determining at acidic pH < 7.5, whereas the release of phosphate from the non-covalent enzyme–phosphate complex (E·P i ) ( k 4 , Scheme ) becomes rate-limiting under basic conditions (pH > 7.5). − In contrast, the phosphorylation of the enzyme ( k 2 ) has been proposed to be the rate-determining step for the reaction of alkyl phosphates …”

Specificity and Kinetics of Haloalkane Dehalogenase

“…Both binding and release of neutral substrates and products were fast. A slow release of a charged product (PO 4 Ϫ , "sticky acid") from the noncovalent complex also occurs in alkaline phosphatase (19), although in this study no evidence was found that release was preceded by an enzyme isomerization, as was suggested by others (20).…”

Room Temperature Phosphorescence Study of Phosphate Binding in Escherichia Coli Alkaline Phosphatase

“…The dimeric AP molecule has a molecular mass of 95 kDa with two tightly bound zinc ions, and one magnesium ion in each of the active centers of the two identical subunits. Its kinetic properties have been well characterized and it has been established that phosphate dissociation from the enzyme is the rate-limiting step in catalysis at alkaline pH (Reid and Wilson, 1971 ;Hull et al, 1976;Bloch and Gorby, 1980;Gettins and Coleman, 1984;Gettins et al, 1985).…”