Escherichia coli alkaline phosphatase. An analysis of transient kinetics

Abstract

1. The hydrolysis of 2,4-dinitrophenyl phosphate by Escherichia coli alkaline phosphatase at pH5.5 was studied by the stopped-flow technique. The rate of production of 2,4-dinitrophenol was measured both in reactions with substrate in excess of enzyme and in single turnovers with excess of enzyme over substrate. It was found that the step that determined the rate of the transient phase of this reaction was an isomerization of the enzyme occurring before substrate binding. 2. No difference was observed between the reaction after mixing a pre-equilibrium mixture of alkaline phosphatase and inorganic phosphate, with 2,4-dinitrophenyl phosphate at pH5.5 in the stopped-flow apparatus, and the control reaction in which inorganic phosphate was pre-equilibrated with the substrate. Since dephosphorylation is the rate-limiting step of the complete turnover at pH5.5, this observation suggests that alkaline phosphatase can bind two different ligands simultaneously, one at each of the active sites on the dimeric enzyme, even though only one site is catalytically active at any given time. 3. Kinetic methods are outlined for the distinction between two pathways of substrate binding, which include an isomerization either of the free enzyme or of the enzyme-substrate complex.

Distinguishing induced fit from conformational selection

“…(ii) a rate limiting conformational change is reflected in kinetic data as a hyperbolic dependence, and (iii) sufficiently high concentrations of protein can be achieved, it is possible to distinguish induced fit from conformational selection [20][21][22][23][24][25][26]. ) is not k 1 +k 2 , which is expected from a slow conformational change followed by fast binding [40].…”

“…An early example was published by Halford already in in 1971 [20]. He demonstrated that the alkaline phosphatase-catalyzed hydrolysis of 2,4-dinitrophenyl phosphate involves a conformational change in the free enzyme.…”

“…1). In this section we complement these studies by describing a straightforward approach, which was previously used by for example Halford [20,29], Olson et al…”

“…It is our hope that the present paper will spur interest in this kinetic method which, while known since many years [20][21][22][23][24][25][26], has been rarely used, has never been explicitly analyzed in a dedicated paper and discussed together with its advantages and limitations.…”

An integrated perspective on RNA aptamer ligand-recognition models: clearing muddy waters

“…In ensemble measurements, it is common to extend this microscopic rate dependence to the effective reaction rate, k eff , which behaves similarly in single-step reactions, and assign IF or CS accordingly. Alkaline phosphatase 47 and adenylate kinase 48 have been classified as CS in this way. Glucokinase has been studied multiple times, and conflicting results indicated that it could bind glucose using either mechanism.…”