Riboflavin synthetase from yeast. Properties of complexes of the enzyme with lumazine derivatives and riboflavin.

Abstract A general mechanism for the riboflavin synthetase reaction involving the addition of 6,7-dimethyl-8-ribityllumazine at two sites on the enzyme has been proposed. One site binds the substrate in such a way that it functions as donor of the 4-carbon moiety transferred in the riboflavin synthetase reaction, while the other binds the lumazine that serves as acceptor of the 4-carbon fragment. The rate equation derived from such a reaction mechanism contains a term second order with respect to the concentration of 6,7-dimethyl-8-ribityllumazine, indicating a possible nonlinear relationship between the reciprocals of velocity and 6,7-dimethyl-8-ribityllumazine concentration. However, zero to first order kinetics were observed over a range of substrate concentration from 2 x 10-4 m to 6 x 10-7 m (compared to 1.0 x 10-5 m for the Michaelis constant) suggesting that binding of the 2 molecules of the lumazine to the enzyme occurs with widely different affinities. Riboflavin synthetase also catalyzes the conversion of 6,7-dimethyl-8-(5'-deoxyribityl)lumazine to 5'-deoxyriboflavin at a slow rate. However, rapid formation of riboflavin-14C (but not 5'-deoxyriboflavin-14C) occurs in the presence of a mixture of equimolar amounts of 6,7-dimethyl-8-ribityllumazine and 6,7-dimethyl-14C-8-(5'-deoxyribityl)lumazine. This implies that 6,7-dimethyl-8-(5'-deoxyribityl)lumazine is an efficient donor of the 4-carbon fragment involved in the formation of the o-xylene portion of riboflavin, but functions poorly as an acceptor of the 4-carbon moiety. Riboflavin synthetase, purified 2000-fold from an extract of bakers' yeast, forms isolatable complexes with substrate (6,7-dimethyl-8-ribityllumazine or 6,7-dimethyl-8-(5'-deoxyribityl)lumazine), riboflavin, and certain inhibitory substrate analogues (e.g. 6-methyl-7-hydroxy-8-ribityllumazine or 6,7-dimethyl-8-d-xylityllumazine). Only those substances are bound which have a kinetic effect on the enzyme; e.g. 6,7-dimethyl-8-l-xylityllumazine does not inhibit activity and is not bound. The complexes contain riboflavin, substrate, or analogues of 6,7-dimethyl-8-ribityllumazine, respectively, in equivalent molar amounts. The binding is not due to covalent bonding since riboflavin can be removed from the protein with charcoal; the treated enzyme retains activity. Furthermore, a tightly bound compound can be rapidly and completely displaced by an excess of the same or another substance with which the enzyme can combine. Thus, displacement has been shown of bound riboflavin by the inhibitory substrate analogue 6-methyl-7-hydroxy-8-ribityllumazine and of bound 6,7-dimethyl-14C-8-ribityllumazine by unlabeled 6,7-dimethyl-8-ribityllumazine. The substrates are probably bound in the complex at the site which leads to donation of the 4-carbon moiety since reaction of equivalent amounts of enzyme-6,7-dimethyl-14C-8-(5'-deoxyribityl)lumazine with 6,7-dimethyl-8-ribityllumazine yields riboflavin-14C only, and not 5'-deoxyriboflavin-14C. Titration of enzyme-6,7-dimethyl-8-ribityllumazine complex with free 6,7-dimethyl-8-ribityllumazine resulted in further polarization of fluorescence. It was calculated that maximal polarization of fluorescence occurred upon the addition of 1 additional eq of substrate to the complex. These results suggest the occurrence of a second substrate-binding site on the enzyme which is involved in the acceptance of the 4-carbon moiety. Attempts to identify an intermediate of the riboflavin synthetase reaction were unsuccessful. No evidence could be obtained that the enzyme requires an organic cofactor for activity.