Characterization of human wild-type and mutant argininosuccinate synthetase proteins expressed in bacterial cells.

Abstract

Argininosuccinate synthetase (ASS) is a urea cycle enzyme with a tetrameric structure composed of identical subunits. Citrullinemia is an autosomal recessive disease caused by a deficiency of ASS. We have previously identified 20 mutations in ASS mRNA of human classical citrullinemia. However, it is difficult to evaluate the effects of each mutation on the enzyme structure and function, since most of the patients are compound heterozygotes. In the present study, wild-type ASS and 12 mutant ASSs were expressed with a bacterial expression system and analyzed enzymologically and immunochemically. The properties of the purified recombinant protein with wild-type human ASS showed good agreement with native enzyme purified from human liver. Mutant ASS proteins with an expected molecular mass, except for delta 7b/Ex16, were highly expressed 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 mutant proteins were as follows: G280R mutant was extracted with an amount of ASS protein similar to wild-type but with no ASS activity, and A192V, 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 Km values for citrulline were obtained in mutant ASSs with A192V (15 mmol/1), R272C (4.2 mmol/l) and R304W. (190 mmol/l) than in wild-type ASS (0.056 mmol/l). The results confirm that these mutations are responsible for ASS deficiency and also indicate that these amino acid residues are important for the function and structure of ASS protein.