Kinetics of the glucose 6-phosphate-glucose exchange activity and glucose inhibition of glucose 6-phosphatase of intact and disrupted rat liver microsomes.

1. The steady state kinetics of the glucose 6-phosphate-glucose exchange catalyzed by glucose 6-phosphatase was determined for intact and disrupted rat liver microsomes. An assay for the exchange activity was devised that entailed measurements of the influences of added glucose on the rates of formation of inorganic phosphate and [14C]glucose from [14C]glucose 6-phosphate. Values obtained with this assay were in excellent agreement with those obtained by measurements of the rates of incorporation of [14C]glucose into glucose 6-phosphate. Activity-pH profiles for the exchange activity were markedly similar to those for the glucose 6-phosphate phosphohydrolase activity. In contrast to other glucose phosphotransferase activities, the exchange activity of untreated microsomes was manifest over a broad range of pH, including neutrality, and the pH optimum (pH 6) was unaffected by disrupting the microsomal structure. With both intact and disrupted microsomes the maximal rates of exchange were greater than the maximal rates of glucose 6-phosphate hydrolysis indicating that the hydrolysis of the phosphoryl-enzyme is the slow step in the phosphohydrolase reaction. 2. Lineweaver-Burk plots describing glucose inhibition of glucose 6-phosphate hydrolysis of intact structures were unlike any classical type of inhibition. As the concentration of glucose was raised the maximal rate of hydrolysis and the apparent Michaelis constant were decreased and the slopes of the lines increased slightly (i.e. the family of plots intersected at a point within the third quadrant). In contrast, the double reciprocal plots describing glucose inhibition of the enzyme of disrupted microsomes intersected at a common point within the second quadrant. The coordinates of the intersection point defined the kinetic constants for the exchange activity. Conversely, primary plots of data for the exchange activity of disrupted microsomes intersected at points within the third quadrant. The coordinates of these intersection points defined the kinetic constants for the hydrolase activity. The value of the inhibitor constant for glucose was greatest with intact microsomes. This could not be attributed to a lesser sensitivity or a decreased accessibility of the enzyme of intact structures to the hexose. 3. The results of this and other recent studies were analyzed in the light of the proposal by Lueck, Herrman and Nordlie ((1972) Biochemistry 11, 2792–2799) that the differences in catalytic properties of the enzyme from untreated and disrupted microsomes can be attributed to detergent- or pH-induced modifications of the individual rate constants associated with the terminal steps of the phosphohydrolase and phosphotransferase activities. This proposal was found to be untenable because it fails to account for (a) the observed changes in the Michaelis constant for glucose 6-phosphate in the phosphohydrolase reaction and (b) the quantitative differences in the responses to detergent supplementation of various phosphotransferase activities of the enzyme.