Temperature-dependent presteady state kinetics of lumazine synthase from the hyperthermophilic eubacterium Aquifex aeolicus.
6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Presteady state kinetic experiments using the enzyme from the hyperthermophilic bacterium Aquifex aeolicus were monitored by multiwavelength photometry. An early optical transient absorbing around 330 nm is interpreted as a Schiff base intermediate obtained by reaction of the position 5 amino group of the heterocyclic substrate with the carbonyl group of 3,4-dihydroxy-2-butanone 4-phosphate. A second transient with an absorption maximum at 445 nm represents an intermediate resulting from the elimination of orthophosphate from the Schiff base. The rate-determining step is the subsequent formation of the 7-exomethylene type anion of 6,7-dimethyl-8-ribityllumazine. The rate constants for the three partial reactions identified by the stopped flow experiments show linear Arrhenius relations in the temperature range of 15-70 degrees C.
Haase
Ilka
I
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany.
Fischer
Markus
M
Bacher
Adelbert
A
Schramek
Nicholas
N
D005051:Eubacterium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D012256:Riboflavin / Q000096:biosynthesis; D013696:Temperature; D013816:Thermodynamics
D009097:Multienzyme Complexes; C044822:6,7-dimethyl-8-ribityllumazine synthase; D012256:Riboflavin