Serine hydroxymethylase. Specificity of bond cleavage to form quinonoid intermediates and rate of holoenzyme formation

Abstract

l-Serine transhydroxymethylase (5,10-methylenetetrahydrofolate:glycine hydroxymethyltransferase, EC 2.1.2.1) a pyridoxal phosphate-dependent enzyme, has been obtained as a homogeneous preparation with a specific activity of 6.7 μmol benzaldehyde per minute at 30°C at pH 7.5 in N-2- hydroxyethylpiperazine-N′-2- ethanesulfonic acid (Hepes) buffer, with dl-threo-β-phenylserine as a substrate. This enzyme has been used to study the specificity of bond cleavage in forming quinonoid intermediates from dl and non-asymmetric amino acids. The ability of the generated quinonoids to react with formaldehyde and acetaldehyde has also been studied and evidence obtained for formation of the corresponding β-hydroxymethyl and β-hydroxyethyl amino acid derivaties. Apotranshydroxymethylase has been prepared and the rate of holoenzyme formation was found to be 0.52 min −1 by measuring Schiff base formation at 425 nm and 0.66 min −1 as determined from restoration of enzymic activity. A requirement for the presence of mercaptoethanol for complete reactivation was also established by these studies.