Studies on alkaline phosphatase. Transient-state and steady-state kinetics of Escherichia coli alkaline phosphatase.
1. The transient-state and steady-state phases of the reaction between Escherichia coli alkaline phosphatase and 4-methylumbelliferyl phosphate were investigated by using a fluorimetric stopped-flow technique. 2. At low substrate concentration (5mum) in the pH range 3.8-6.3 there was an initial rapid liberation of up to 1mole of 4-methylumbelliferone/mole of enzyme. 3. At very low substrate concentration (0.1mum) in the pH range 4.9-5.9 an initial lag in 4-methylumbelliferone production was observed, from which values for k(+1) and k(-1) could be obtained. 4. The pH profiles for the rates of phosphorylation and dephosphorylation are quite different, and it is postulated that an ionizing group which determines the conformation during the phosphorylation step is not involved in the dephosphorylation step. 5. The binding constants for substrate and P(i) are similar throughout the pH range 4-8. The ionization of substrate or P(i) appeared to have no marked effect on the binding.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D001665:Binding Sites; D003374:Coumarins / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics
D003374:Coumarins; D000469:Alkaline Phosphatase