The Kinetics of Phosphoglucoisomerase

Abstract

Phosphoglucoisomerase(isomerase) is present in most tissues at very high levels of activity.Because of its strategic position in relation to alternate metabolic pathways, its kinetics are of interest.The kinetics are also of practical concern in connection with the use of glucose 6phosphate to measure enzyme systems which produce glucose 6-phosphate, since isomerase, an ubiquitous contaminant, could distort the results if it diverted some of the product to fructose 6phosphate.Studies are described with partially purified isomerase from rabbit brain and muscle, and from human erythrocytes.Assay conditions were designed to prevent accumulation of the products and thereby minimize product inhibition and back reactions.The substrates consisted of enzymatically analyzed glucose-6-P and enzymatically prepared and analyzed fructose-6-P.Contaminants of chemically prepared fructose-6-P appear to have confused earlier studies and led to faulty values for the equilibrium ratio between the isomeric hexose phosphates.The Michaelis constants have been found to be very small in both directions, but the constant for fructose-6-P is much the smaller.This results from the fact that the reaction velocity with substrate excess is approximately the same in both directions, even though equilibrium substantially favors glucose-6-P formation. MATERIALSUnless otherwise noted homogenates were prepared in a Waring Blendor, and all enzyme fractionations were carried out at 0 to 4".Salt fractionations were made at a neutral pH by the addition of 1 mole of ammonium hydroxide per 50 moles of ammonium sulfate.Precipitates were removed by centrifuging at 13,000 to 16,000 X g for 10 to 15 minutes.Muscle Isomeruse-Rabbit muscle was homogenized with 5 volumes of water with the addition of sufficient 1 N NH,OH to maintain the pH near 7. Material which sedimentcd at 8,000 X g in 10 minutes was discarded.The fraction soluble in 2 M ammonium sulfate and insoluble in 3 M was refractionated three times with ammonium sulfate between 1.2 and 2.3 M, 2.0 and 2.4 M, and 1.5 and 2.4 M at respective volumes of 0.34, 0.40, and 0.14 ml per g of original muscle.The yield was 28% of the original activity with 14-fold purification.Activity was 2190 moles per kg of protein per hour at 38" (measured in the direction glucose-6-P to fructose-6-P).This is comparable to the activity of a preparation described by Slein (1).The enzyme is stable for at least several weeks if kept frozen at pH 7 to 8.