Biosynthesis of riboflavin single turnover kinetic analysis of 6,7-dimethyl-8-ribityliumazine synthase

被引:18
作者
Schramek, N [1 ]
Haase, I [1 ]
Fischer, M [1 ]
Bacher, A [1 ]
机构
[1] Tech Univ Munich, Lehrstuhl Organ Chem & Biochem, D-85747 Garching, Germany
关键词
D O I
10.1021/ja028226k
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4-(1 H,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) muM(-1). Rapid mixing of the complex between enzyme and pyrimicline 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.
引用
收藏
页码:4460 / 4466
页数:7
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