KINETIC-ANALYSIS OF THE ACTIVATION OF ZYMOMONAS-MOBILIS GLUCOKINASE BY PHOSPHATE

A detailed kinetic analysis of glucokinase EC 2.7.1.2 from Zymomoras m obilis has been carried out. This enzyme has an absolute requirement f or inorganic phosphate as activator, and the kinetic behaviour can be interpreted as a steady-state ordered mechanism in which glucose is th e first substrate. Values for each of the kinetic constants have been obtained for the conditions I = 0.12, 30 degrees C, and pH 7.0. Direct binding studies have confirmed that ATP does not bind to the enzyme w ithout glucose present. Phosphate does not affect ATP binding to the e nzyme-glucose complex; when saturated with both ATP and glucose, the d issociation constant for phosphate (determined kinetically) is 0.045 m M. When saturated with the other substrate and phosphate, the K-m valu es for glucose and MgATP are 0.095 mM and 0.19 mM, respectively. The i onic form of phosphate is not important, as the apparent K-m for phosp hate did not change significantly over the pH range 6.4 to 7.5. Raisin g the temperature increased V-max at the high rate of 10% per degree, which correlates well with the fermentation rates between 20 and 30 de grees C, giving further support to the concept that glucokinase is the rate-controlling enzyme in Z. mobilis glucose fermentation.