CHARACTERIZATION OF HUMAN WILD-TYPE AND MUTANT ARGININOSUCCINATE SYNTHETASE PROTEINS EXPRESSED IN BACTERIAL-CELLS

Argininosuccinate synthetase (ASS) is a urea cycle enzyme with a tetra meric structure composed of identical subunits. Citrullinemia is an au tosomal recessive disease caused by a deficiency of ASS. We have previ ously identified 20 mutations in ASS mRNA of human classical citrullin emia. However, it is difficult to evaluate the effects of each mutatio n on the enzyme structure and function, since most of the patients are compound heterozygotes. In the present study, wild-type ASS and 12 mu tant ASSs were expressed with a bacterial expression system and analyz ed enzymologically and immunochemically. The properties of the purifie d recombinant protein with wild-type human ASS showed good agreement w ith native enzyme purified from human liver. Mutant ASS proteins with an expected molecular mass, except for Delta 7b/Ex16, were highly expr essed in the bacterial cells. It was difficult to extract ASS proteins with some mutations (A118T, Delta Ex7, R157H, R363W, R363L, G390R and ins37b/Ex15&16) from cells by freezing and thawing. Extractable mutan t proteins were as follows: G280R mutant was extracted with an amount of ASS protein similar to wild-type but with no ASS activity, and A192 V, R272C and R304W mutants detected various amounts of ASS protein (13 , 110 and 33% of wild-type, respectively) with a low ASS activity and abnormal kinetics. Higher K-m values for citrulline were obtained in m utant ASSs with A192V (15 mmol/l), R272C (4.2 mmol/l) and R304W (190 m mol/l) than in wild-type ASS (0.056 mmol/l). The results confirm that these mutations are responsible for ASS deficiency acid also indicate that these amino acid residues are important for the function and stru cture of ASS protein.