Restructuring the active site of fumarase for the fumarate to malate reaction

Abstract

Changes in the active site of fumarase (yeast fumarase II) that occur when fumarate is converted to malate (E.F --> E.M) must be reversed for another cycle of reaction to take place. As shown here, recycling of the enzyme includes two proton transfers and one conformational change. These events, together with the M-off step, are variously rate-determining depending on the medium. In very low salt the release of M is limited by the conformational change. Thus, (V/Km)F decreases with increased viscosity, shown with glycerol. A variety of simple anions, such as Cl- at approximately 50 mM and F itself at low concentration, activate the dissociation of M. This nonspecific anion effect is the basis for the >4-fold apparent cooperative activation by substrate. The M-off step and the conformational change are independent and random-order events. Thus, even when M-off is made rapid the rate of recycling is inhibited by glycerol, which in 100 mM NaCl inhibits Vmax but not V/Km. The enzyme form that results when M is released is M-specific, Em. Thus mesotartarate, competitive toward M, is noncompetitive toward F. The slow conformational change required for recycling of Em is activated by Pi and chaotropic anions such as azide and thiocyanate, giving rise to a nonspecific intermediate, Emf (mesotartarate becomes competitive toward F and Britton's countertransport property disappears with these activators). Evidence is presented for the locations and rates of the two proton transfer steps required to complete the cycle.