Argininosuccinate synthetase activity in cultured human lymphocytes

The activity of argininosuccinate synthetase (E.C. 6.3.4.5), a urea cycle enzyme, was measured in cultured human lymphocytes using a new radioactive assay. Control cells had a maximum specific activity of 15.7±8.7 nmoles per hour per milligram of protein and an apparent K m for citrulline of 2 × 10 −4 m , whereas cells derived from a patient with citrullinemia had no detectable activity. A nutritional variant, selected out of the citrullinemic lymphocyte population by ability to grow in citrulline, had a maximum specific activity of 10.7±3.8 nmoles/hr/mg and an apparent K m for citrulline of 2 × 10 −2 m . These measurements confirm the observation that citrullinemia is associated with a defect in argininosuccinate synthetase activity and provide further evidence that citrullinemia is expressed in cultured lymphocytes. The emergence of a nutritional variant with a partial defect in argininosuccinate synthetase enzyme suggests that this citrullinemic patient has a heterogeneous population of cells, some totally defective and others only partially defective in argininosuccinate synthetase. The new activity assay is described in detail.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44125/1/10528_2004_Article_BF00484469.pd

Enzymatic Reaction Mechanisms, pp ranca

ABSTRACT: The mechanism of the argininosuccinate lyase reaction has been probed by the measurement of the effects of isotopic substitution a t the reaction centers. A primary deuterium isotope effect of 1.0 on both Vand V / K is obtained with (2S,3R)-argininosuccinate-3-d, while a primary 15N isotope effect on V / K of 0.9964 f 0.0003 is observed. The 15N isotope effect on the equilibrium constant is 1.018 f 0.001. The proton that is abstracted from C-3 of argininosuccinate is unable to exchange with the solvent from the enzyme-intermediate complex but is rapidly exchanged with solvent from the enzyme-fumaratearginine complex. A deuterium solvent isotope effect of 2.0 is observed on the V,,, of the forward reaction. These and other data have been interpreted to suggest that argininosuccinate lyase catalyzes the cleavage of argininosuccinate via a carbanion intermediate. The proton abstraction step is not rate limiting, but the inverse 15N primary isotope effect and the solvent deuterium isotope effect suggest that protonation of the guanidino group and carbon-nitrogen bond cleavage of argininosuccinate are kinetically significant. Argininsuccinate lyase catalyzes the cleavage of argininosuccinate to arginine and fumarate. The enzyme is found in the liver where it functions in the biosynthesis of urea. The enzyme from bovine liver has been shown by Lusty and Ratner (1972) to be a tetramer of four identical subunits. No external cofactor is involved, and the enzyme apparently does not require metal ions for catalytic activity. The details of the catalytic events leading to the chemical transformation of argininosuccinate to fumarate and arginine are largely unknown. Ratner and co-workers have shown that the reaction involves the trans elimination of arginine and the pro-R hydrogen at C-3 of argininosuccinate (Hoberman et al., 1965). The kinetic mechanism of the reaction is random In this paper we report on our efforts to determine the magnitude and the timing of the bond-breaking steps in the conversion of argininosuccinate to arginine and fumarate. Th