Pre-steady-state kinetic analysis of riboflavin synthase using a pentacyclic reaction intermediate as substrate.
Riboflavin synthase catalyses a mechanistically complex dismutation affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H )-pyrimidinedione from 6,7-dimethyl-8-ribityllumazine. A pentacyclic adduct (compound 2 ) of two substrate molecules was used as substrate for pre-steady-state kinetic analysis. Whereas the wild-type enzyme catalyses the decomposition of compound 2 into a mixture of riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H )-pyrimidinedione, as well as into two equivalents of 6,7-dimethyl-8-ribityllumazine, a H102Q mutant enzyme predominantly catalyses the former reaction. Stopped-flow experiments with this mutant enzyme failed to identify a reaction intermediate between compound 2 and riboflavin. However, the apparent rate constants for the formation of riboflavin as observed by stopped-flow and quenched-flow experiments were significantly different, thus suggesting that the reaction proceeds via a significantly populated intermediate, the absorbance of which is similar to that of compound 2 . An F2A mutant enzyme converts compound 2 predominantly into 6,7-dimethyl-8-ribityllumazine. Stopped-flow experiments using compound 2 as substrate indicated a slight and rapid initial increase in absorbance at 310 nm, followed by a slower decrease. This finding, in conjunction with different apparent rates for the formation of 6,7-dimethyl-8-ribityllumazine, suggests the involvement of a significantly populated intermediate in the transition between compound 2 and 6,7-dimethyl-8-ribityllumazine, the optical spectrum of which is similar to that of compound 1.
Illarionov
Boris
B
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
Haase
Ilka
I
Fischer
Markus
M
Bacher
Adelbert
A
Schramek
Nicholas
N
D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009154:Mutation; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism; D012258:Riboflavin Synthase / Q000235:genetics / Q000378:metabolism*
D011621:Pteridines; C001061:6,7-dimethyl-8-ribityllumazine; D012258:Riboflavin Synthase