Stopped-flow studies TMADH

Over the years there have been a number of mechanistic proposals for substrate oxidation by TMADH. An early proposal considered a carbanion mechanism in which an active site base deprotonates a substrate methyl group to form a substrate carbanion [69] reduction of the flavin was then achieved by the formation of a carbanion-flavin N5 adduct, with subsequent formation of the product imine and dihydroflavin. A number of active site residues were identified as potential bases in such a reaction mechanism. Directed mutagenesis and stopped-flow kinetic studies, however, have been used to systematically eliminate the participation of these residues in a carbanion-type mechanism [76-79], thus indicating that a proton abstraction mechanism initiated by an active site residue does not occur in TMADH. Early proposals also invoked the trimethylammonium cation as the reactive species in the enzyme-substrate complex, owing to the high (9.81) of free... 

Substrate bond breakage by wild-type TMADH is too fast to be followed using the stopped-flow method in the regime where both His-172 and trimethylamine in the enzyme-substrate complex are deprotonated (that is pH 10). Consequently, our tunneling studies have focused on the compromised mutant enzymes H172Q and Y169F ( 10-fold and 40-fold reduction in the limiting rate constant for flavin reduction compared with wild-type enzyme, respectively). 

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