Kinetics of the Escherichia coli alkaline phosphatase catalyzed hydrolysis of 2,4-dinitrophenyl phosphate

Ko, Sai-Hang D.; Kézdy, Ferenc J.

doi:10.1021/ja01002a068

Cited by 54 publications

(28 citation statements)

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“…A 1:1 stoicheiometry was observed between the concentration of 2,4-dinitrophenol and the concentration of active enzyme in the zincdeficient enzyme as measured above. The 1:1 stoicheiometry agrees with the findings of Ko & K6zdy (1967) and Trentham & Gutfreund (1968).…”

Section: Methods and Resultssupporting

confidence: 89%

A substrate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli

Halford

Bennett

Trentham

et al. 1969

Biochemical Journal

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1. Benzyl phosphonates were prepared and their potentialities as chromophoric reagents for the exploration of the substrate-binding site of Escherichia coli alkaline phosphatase were investigated. 4-Nitrobenzylphosphonate is a competitive inhibitor of the enzyme. 2-Hydroxy-5-nitrobenzylphosphonate changes its spectrum on binding to the enzyme. This spectral change is reversed when the phosphonate is displaced from the enzyme by substrate. 2. The kinetics of the reaction of 2-hydroxy-5-nitrophenylphosphonate were studied by the stopped-flow and the temperature-jump techniques. It was found that the combination of the phosphonate with the enzyme occurred in two successive and reversible steps: enzyme-phosphonate complex-formation followed by rearrangement of the complex. The spectral change is associated with the rearrangement. At pH8 in 1m-sodium chloride at 22 degrees the rate constant is 167sec.(-1) for the rearrangement of the initially formed binary complex and is 18sec.(-1) for the reverse process. 3. It has previously been proposed that the reactions of phosphatase with its substrates include a distinct step between enzyme-substrate combination and chemical catalysis. The rate constant involved could be predicted but not measured from experiments with substrates. The value for the rate constant measured from the rate of the enzyme-phosphonate rearrangement is in excellent agreement with the predicted value. A model for the reaction mechanism is proposed that includes a conformation change in response to phosphate ester binding before phosphate transfer from substrate to enzyme.

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Section: Methods and Resultssupporting

confidence: 89%

A substrate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli

Halford

Bennett

Trentham

et al. 1969

Biochemical Journal

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“…The reactions of dimeric alkaline phosphatase with excess of substrates at acidic pH values have been studied by Fernley & Walker (1966, 1968), Ko & Kezdy (1967) and Trentham & Gutfreund (1968). The observations on this reaction presented here confirm their findings, but an alternative interpretation is developed fronm them.…”

Section: Discussionsupporting

confidence: 76%

“…The transient phase of substrate hydrolysis by alkaline phosphatase was studied in the presence of the competitive inhibitor Pi. The decrease in the rate constant of transient product formation, without alteration to its amplitude, is the expected effect of a competitive inhibitor on this reaction provided that the enzyme-inhibitor complex is in rapid equilibrium with free enzyme and inhibitor (Ko & Kezdy, 1967). But the existence of the transient phase during substrate hydrolysis indicates that dephosphorylation limits the catalytic-centre activity at acidic pH values (Trentham & Gutfreund, 1968) presenting further identification of this process as the rate-determining step under acidic conditions.…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli alkaline phosphatase. An analysis of transient kinetics

Halford

1971

Biochemical Journal

100

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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.

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“…The rate constant for the transient phase of hydrolysis of this: substrate by cobalt phosphatase is very rapid, with a half-time of about 15 msec, even though the low temperature and low ionic strength represent suboptimal conditions. Similar values of the rate constant for phosphorylation of the enzyme by substrate for the zinc protein, estimated from the data of Fernley and Walker (1966) and KO and Kezdy (1967), indicates that this rate constant for the cobalt protein is nearly ten times greater than that expected for zinc phosphatase. The present data demonstrate that dephosphorylation is rate limiting for the hydrolysis of phosphomonoesters by the cobalt enzyme at pH 8.…”

Section: Kinetics Of Cobalt Alkaline Phosphatasesupporting

confidence: 54%

“…The mechanism of the nonspecific phosphatases has been shown to involve a particularly reactive seryl residue that is phosphorylated by inorganic phosphate at low pH or during the course of enzymatic activity by substrate at neutral pH (Schwartz & Lippman, 1961;Engstrom, 1962). Careful kinetic studies of the steady-state hydrolysis of 4-NPPt by Lazdunski and Lazdunski (1966), and of the presteady-state hydrolysis of 5-MeUP,t 2-NPPt and 2,4-DNPPt by various workers (Fernley & Walker, 1966;Fife, 1966;KO & Kezdy, 1967) have led to findings summarized in TABLE 4. These must be considered for inclusion in any kinetic scheme devised for alkaline phosphatase.…”

Section: Kinetics Of Cobalt Alkaline Phosphatasementioning

confidence: 99%