Biosynthesis of riboflavin. Single turnover kinetic analysis of 6,7-dimethyl-8-ribityllumazine synthase.
6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate, affording the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine. Single turnover experiments monitored by multiwavelength photometry were performed with the recombinant lumazine synthase of Bacillus subtilis. Mixing of the enzyme with the pyrimidine type substrate is conducive to a hypsochromic shift as well as a decrease in absorbance of the heterocyclic substrate; the rate constant for that reaction is 0.02 s(-1) microM(-1). Rapid mixing of the complex between enzyme and pyrimidine type substrate with the second substrate, 3,4-dihydroxy-2-butanone 4-phosphate, is followed by the appearance of an early optical transient characterized by an absorption maxima at 330 nm of low intensity which was tentatively assigned as a Schiff base intermediate. The subsequent elimination of phosphate affords a transient with intense absorption maxima at 455 and 282 nm, suggesting an intermediate with an extended system of conjugated double bonds. The subsequent formation of the enzyme product, 6,7-dimethyl-8-ribityllumazine, is the rate-determining step.
Schramek
Nicholas
N
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
Haase
Ilka
I
Fischer
Markus
M
Bacher
Adelbert
A
D001412:Bacillus subtilis / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011621:Pteridines / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012256:Riboflavin / Q000096:biosynthesis*; D013056:Spectrophotometry, Ultraviolet
D009097:Multienzyme Complexes; D011621:Pteridines; D011994:Recombinant Proteins; C001061:6,7-dimethyl-8-ribityllumazine; C044822:6,7-dimethyl-8-ribityllumazine synthase; D012256:Riboflavin