Physicochemical Characterization of Citrate Synthase and Its Subunits

Abstract

Abstract Citrate synthase has a molecular weight of 1.0 x 105, as determined from sedimentation equilibrium and also from a combination of hydrodynamic properties. The sedimentation and diffusion coefficients were found to be 6.0 S and 5.8 x 10-7 cm2 sec-1, respectively, and the intrinsic viscosity was 3.95 ml g-1. Optical rotatory dispersion showed a 233 mµ trough and a 198 mµ peak with [m']233 = -7,150 and [m']198 = +28,500 deg cm2 dmole-1; the b0 of the Moffitt equation was -240. Circular dichroism showed a double minimum at 222 and 210 mµ and a maximum at 191 mµ with [θ]222 = -20,000, [θ]210 = -21,000, and [θ]191 = +42,000 deg cm2 dmole-1. These findings indicate the presence of a moderate amount of α-helical segments in the protein molecule. Citrate synthase does not dissociate in the presence of 2 m KCl, dioxane, 8 m urea, p-chloromercuribenzoate, or β-mercaptoethanol, but it can be readily split into two physically indistinguishable subunits upon succinylation. The succinylated subunit was enzymatically inactive. Its sedimentation coefficient was one-half that of the native enzyme and its diffusion coefficient was reduced by about 10%. The optical properties also suggested a helical content of about one-half that of the native protein. Citrate synthase can also dissociate into two subunits in 6 m guanidine hydrochloride, but the molecule becomes completely unordered.