A substate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli.
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.
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors; D001665:Binding Sites; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008956:Models, Chemical; D063065:Organophosphonates; D013053:Spectrophotometry
D063065:Organophosphonates; D000469:Alkaline Phosphatase