Further studies on aspartate transcarbamoylase: molecular weight of the c3r6 complex and analysis of succinate inhibition in the native enzyme

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Abstract
The complex which is formed when excess regulatory subunits (r2) of aspartate transcarbamoylase (EC 2.1.3.2) [of Escherichia coli] are added to a dilute solution of the catalytic subunit (c3) was studied by gel-filtration on Sephadex G-200. The elution volume indicates a Stokes'' radius of between 5.42-5.92 nm, depending on the method of calculation. Using the sedimentation coefficient of 7.7 S previously determined, the MW is .apprx. 200,000, in support of the c3r6 structure proposed earlier for the complex. The calculated frictional coefficient indicates abnormal hydrodynamic properties which are probably due to unusual structure characteristics. The pattern of succinate inhibition of native aspartate transcarbamoylase was also analyzed. At low concentrations, succinate activates the enzyme, presumably by converting it from the taut state to the relaxed (R) state. Further increase in the succinate concentration leads to competitive inhibition of the R state. Using a novel procedure for analysis of the data, the Km for aspartate of the R state was .apprx. 7 mM. This value is close to the Km of c3r6 for aspartate, measured under identical conditions. The c3r6 complex apparently resembles the R state of the native enzyme.