Study on the initial kinetics of yeast phosphofructokinase by stopped-flow measurements.
The initial kinetics of yeast phosphofructokinase was studied by stopped-flow measurements over an enzyme concentration range from 0.5 mg/ml to 0.01 mg/ml. Before attaining the steady state the reaction showed a lag phase in the product formation, the duration of which was found to decrease with increasing enzyme concentration. The lag phase disappeared after preincubation of the enzyme for at least five minutes with either fructose 6-phosphate, fructose 1,6-bisphosphate or fructose 2,6-bisphosphate. Preincubation of the enzyme with either AMP or ADP resulted in a reduction of this phase, while ATP was without effect. Simultaneous addition of fructose 1,6-bisphosphate to the reaction mixture of the enzyme causes a significant shortening of the transient phase, whereas micromolar concentrations of fructose 2,6-bisphosphate are capable of abolishing the lag phase completely. The occurrence of an initial transient phase suggests that the enzyme after starting the reaction converts from a state of low activity to one of high activity. This conversion mainly depends on the concentration of fructose 1,6-bisphosphate generated in the course of the reaction. In addition an association reaction of the enzyme seems to be involved in the process of conversion of the phosphofructokinase during the initial transient phase.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005635:Fructosediphosphates / Q000494:pharmacology; D005636:Fructosephosphates / Q000494:pharmacology; D007700:Kinetics; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D005635:Fructosediphosphates; D005636:Fructosephosphates; D000244:Adenosine Diphosphate; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; C029063:fructose-1,6-diphosphate