Product inhibition of the hexokinases.

ADP3and ADPMg caused mixed inhibition of purified yeast hexokinase isoenzymes and ascites tumor hexokinases I and II when ATPMg is the varied substrate. Since exchange between ADPMg and ATPMg is observed under steady state conditions in which free glucose-6-P is not available for reaction, the effect of ADPMg on V,,, must result from its reaction at the ADP product site of the enzyme. glucose-6-P complex. Thus, this type of inhibition, which is rare among the kinases, is attributed to a relatively slow release of the second product, glucose-6-P. In the case of yeast hexokinase, the replot of the intercepts of the l/V against l/ATPMg plot as a function of ADPMg concentration is hyperbolic. Thus, the alternative sequence of product release, glucose-6-P first, is occurring; however, this sequence is probably of minor significance except in the presence of high concentrations of ADPMg. Under certain conditions, mixed inhibition can be observed by glucose-6-P with ATPMg or ITPMg, the variable substrate. In no case was exchange observed between glucose-6-32P and ATPMg under product-inhibited steady state conditions in which free ADP was kept low by a suitable trapping reaction. This indicates that any inhibition of V ma* by glucose-6-P is not due to action at the glucose-6-P product site of an enzyme,ADP complex which would have to dissociate rapidly, but to action at a particular modifier site. Such a site is therefore indicated for tumor hexokinase II. A simple general kinetic procedure is described for evaluating the inhibition due to ADP3when other inhibitors such as ADPMg and ATP4are also present.