| Description |
For several years, scientists who study enzyme catalyzed reaction have suggested that the reaction rates are rarely dependent upon the actual chemistry but rather on protein motion during the conformational change that occurs as products are released from the active site. Yeast hexokinase is an enzymatic protein with two large, moving subunits which surround the; active site where glucose molecules are phosphorylated in the initial step of glycolysis. If subunit movement during product release constituted the rate determining step of the hexokinase transformation, the volume of activation should be very large. The volume of activation for yeast hexokinase was determined by stopped-flow spectrophotometry. The reaction rate of hexokinase was measured at several pressures by measuring the reaction rate; of a coupled reaction system in which the transformation by hexokinase was rate limiting. The phosphorylation of glucose by hexokinase was coupled with the much faster transformation of NAD+ to NADH by glucose 6-phosphate dehydrogenase because the rate of NADH formation could be easily monitored spectrophotometrically at 340 nanometers. The slope of a graph in which the natural logarithms of the mean reaction rates were plotted against pressure was determined to be -(2.99 +/- 0.32) E-04. This value was used to calculate the volume of activation for yeast hexokinase which is (7.4 +/- 0.8) cm3; /mol. Although this result is somewhat smaller than expected, it is consistent with the theory that product release is the rate determining step of enzyme catalyzed reactions. |
