Biosynthesis of Urea: XIII. DISSOCIATION-ASSOCIATION KINETICS AND EQUILIBRIA OF ARGININOSUCCINASE

Abstract

Abstract Further study of the cold inactivation of argininosuccinase shows that a new, more slowly sedimenting component is formed which has an S value of about 5.6 as compared to 9.3 for the active enzyme. The two components can be resolved by high speed sedimentation in phosphate but not in Tris or imidazole buffers. The proportion of each component corresponded to the extent of inactivation. On resolution in a sucrose density gradient, the 5.6 S component was found to be enzymatically inactive. Activity was regained by thermal reactivation of the fractions in this peak. Thus, cold inactivation and thermal reactivation are accompanied by reversible dissociation of the enzyme into two, probably identical, subunits which are one-half the size of the active enzyme (molecular weight 202,000). The rate of dissociation followed first order kinetics and the velocity constants increased with increasing pH. Association followed first order or second order kinetics depending on the buffer. A mechanism for the reversible dissociation has been proposed in the light of the kinetic analysis. The kinetic patterns in the direction of association reveal temperature-dependent equilibria between the dimeric and monomeric forms of the enzyme. The equilibrium constant and the negative change in free energy decreased with a decrease in temperature. The enthalpy and entropy changes were found to be 46 kcal and 189 e.u., respectively, per mole of active enzyme. The negative temperature dependence for association and the large positive value for the entropy change suggests that the active form of the enzyme is stabilized by hydrophobic bonds.