Analysis of progress curves. Interaction of pyruvate kinase from Escherichia coli with fructose 1,6-bisphosphate and calcium ions.
The influence of fructose 1,6-bisphosphate and Ca2+ on the kinetics of pyruvate kinase from Escherichia coli K12 was studied (at pH 7.0 and 25 degrees C) by using the pH-stat method for the measurement of the reaction progress as well as initial-rate analysis. The data were analysed on the basis of a concerted model with three conformational states [Markus, Plesser, Boiteux, Hess & Malcovati (1980) Biochem. J. 189, 421-433] by using a novel procedure for a computer-directed treatment of progress curves [Markus & Plesser (1976) Biochem. Soc. Trans. 4, 361-364]. By addition of fructose 1,6-bisphosphate the sigmoid kinetics with respect to phosphoenolpyruvate and Mg2+ is abolished and the activity of the enzyme is described by classical saturation kinetics. This is explained by exclusive binding of fructose 1,6-bisphosphate at an allosteric site of the conformational state that forms the active complex. We observe that Ca2+ is an activator of the enzyme at low Mg2+ and Ca2+ concentrations; otherwise it is an inhibitor. These effects can be understood by assuming that Ca2+ has the same binding properties as Mg2+, although it does not allow a catalytic turnover.
D002118:Calcium / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005635:Fructosediphosphates / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008433:Mathematics; D011770:Pyruvate Kinase / Q000378:metabolism*
D005635:Fructosediphosphates; D011770:Pyruvate Kinase; D008274:Magnesium; C029063:fructose-1,6-diphosphate; D002118:Calcium