Number of arginine residues in the substrate binding sites of rat liver cystathionase.

It has been reported that rat liver cystathionase (EC 4.4.1 .l) is inhibited by butanedione and suggested that the a-carboxyl groups of the substrates (L-homoserine and L-cysteine, respectively) are bound to the protein through arginine residues [I]. To support the concept that specific residues are critical or even essential to enzyme activities, we carried out experiments using phenylglyoxal, another fairly specific reagent, for the chemical modification of arginine residues in proteins [2]. Two kinds of experiment were carried out : (i) [14~]Phenylglyox~ (CER, Saclay) was used to check whether radioactivity was incorporated into cystathionase. (ii) Phenylglyoxal monohydrate (Aldrich) was used to determine the resulting inhibition of cystathionase activity. Amino acid analysis of hydrolysates of native enzyme and of phenylglyox~-treated enzyme revealed that only the level of arginine was modified by incubation with phenylglyoxal. As this investigation was directed mainly at determining the number of arginine residues involved in substrate binding, S-carboxyethylhomocysteine (S-CH), a competitive inhibitor of cystathionase [3,4], was added to the enzyme before incubation with phenylglyoxal and the number of arginine residues modified under those conditions was determined. A preliminary report of some of these findings has appeared [ 51. 2. Materials and methods