Physicochemical and kinetic properties of beef liver argininosuccinase. Studies in the presence and absence of arginase.

Physicochemical properties of beef liver argininosuccinase (EC 4.3.2.1) are reported, which show that the operational kinetic unit at pH 7.5 in the concentration range used for enzymic assays is of molecular weight 203 000. This information is utilized in the interpretation of initial velocity studies of the forward reaction (argininosuccinate as initial substrate) where, in an Eadie-Hofstee plot, deviation from linearity is observed. These results are examined with the aid of a rate equation, formulated as a ratio of two polynomials in initial substrate concentration, which provides a quantitative description of the system in terms of cooperative effects. The kinetics of the reverse reaction catalysed by argininosuccinase were also investigated by performing experiments of different design in which the rate of change of fumarate concentration was monitored as a function of time. Comparison of these results with numerical solutions obtained by integrating the differential rate equation reflecting the reversible reaction permitted estimation of the relevant kinetic parameters. One of the latter experiments involved a coupled assay with argininosuccinase and arginase as the consecutive catalysts and indicated that no chemical interaction occurred between the enzymes. This observation, which is relevant to consideration of fluxes in the urea cycle, was supported by both sedimentation velocity studies on mixtures of the enzymes and by other kinetic analysis.