Reaction of the C30A mutant of trimethylamine dehydrogenase with diethylmethylamine.
The role played by the 6-S-cysteinyl-FMN bond of trimethylamine dehydrogenase in the reductive half-reaction of the enzyme has been studied by following the reaction of the slow substrate diethylmethylamine with a C30A mutant of the enzyme lacking the covalent flavin attachment to the polypeptide. Removal of the 6-S-cysteinyl-FMN bond diminishes the limiting rate for the first of the three observed kinetic phases of the reaction by a factor of 6, but has no effect on the rate constants for the two subsequent kinetic phases. The flavin in the C30A enzyme recovered from the reaction of the C30A enzyme with excess substrate is found to have been converted to the 6-hydroxy derivative, rendering the enzyme inactive. The noncovalently bound FMN of the C30A mutant enzyme is also converted to 6-hydroxy-FMN and rendered inactive upon reduction with excess trimethylamine, but not by reduction with dithionite, even at high pH or in the presence of the effector tetramethylammonium chloride. These results suggest that one significant role of the 6-S-cysteinyl-FMN bond is to prevent the inactivation of the enzyme during catalysis. A reaction mechanism is proposed whereby OH- attacks C-6 of a flavin-substrate covalent adduct in the course of steady-state turnover to form 6-hydroxy-FMN.
Huang
L
L
Department of Medical Biochemistry, Ohio State University, Columbus, Ohio 43210, USA.
Scrutton
N S
NS
Hille
R
R
D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D004048:Diethylamines / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000201:enzymology / Q000235:genetics; D006900:Hydroxylation; D007700:Kinetics; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D013379:Substrate Specificity
D004048:Diethylamines; C090758:diethylmethylamine; D005486:Flavin Mononucleotide; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; D003545:Cysteine