ROLE OF ARG-401 OF CYTOSOLIC SERINE HYDROXYMETHYLTRANSFERASE IN SUBUNIT ASSEMBLY AND INTERACTION WITH THE SUBSTRATE CARBOXY GROUP

In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltrans ferase, a highly conserved Arg-401 was mutated to Ala by site-directed mutagenesis. The mutant enzyme had a characteristic visible absorbanc e at 425 nm indicative of the presence of bound pyridoxal 5'-phosphate as an internal aldimine with a lysine residue. However, it had only 0 .003% of the catalytic activity of the wild-type enzyme. It was also u nable to perform reactions with glycine, beta-phenylserine or D-alanin e, suggesting that the binding of these substrates to the mutant enzym e was affected. This was also evident from the interaction of amino-ox yacetic acid, which was very dow (8.4 x 10-4 s(-1) at 50 mu M) for the R401A mutant enzyme compared with the wild-type enzyme (44.6 s(-1) at 50 mu M). In contrast, methoxyamine (which lacks the carboxy group) r eacted with the mutant enzyme (1.72 s(-1) at 250 mu M) more rapidly th an the wild-type enzyme (0.2 s(-1) at 250 mu M). Further, both wild-ty pe and the mutant enzymes were capable of forming unique quinonoid int ermediates absorbing at 440 and 464 nm on interaction with thiosemicar bazide, which also does not have a carboxy group. These results implic ate Arg-401 in the binding of the substrate carboxy group. In addition , gel-filtration profiles of the apoenzyme and the reconstituted holoe nzyme of R401A and the wild-type enzyme showed that the mutant enzyme remained in a tetrameric form even when the cofactor had been removed. However, the wildtype enzyme underwent partial dissociation to a dime r, suggesting that the oligomeric structure was rendered more stable b y the mutation of Arg-401. The increased stability of the mutant enzym e was also reflected in the higher apparent melting temperature (T-m) (61 degrees C) than that of the wild-type enzyme (56 degrees C). The a ddition of serine or serinamide did not change the apparent T-m of R40 1A mutant enzyme. These results suggest that the mutant enzyme might b e in a permanently 'open' form and the increased apparent T-m could be due to enhanced subunit interactions.