Reductive half-reaction of the H172Q mutant of trimethylamine dehydrogenase: evidence against a carbanion mechanism and assignment of kinetically influential ionizations in the enzyme-substrate complex.
The reactions of wild-type trimethylamine dehydrogenase (TMADH) and of a His-172-->Gln (H172Q) mutant were studied by rapid-mixing stopped-flow spectroscopy over the pH range 6.0-10.5, to address the potential role of His-172 in abstracting a proton from the substrate in a 'carbanion' mechanism for C-H bond cleavage. The pH-dependence of the limiting rate for flavin reduction (klim) was studied as a function of pH for the wild-type enzyme with perdeuterated trimethylamine as substrate. The use of perdeuterated trimethylamine facilitated the unequivocal identification of two kinetically influential ionizations in the enzyme-substrate complex, with macroscopic pKa values of 6.5+/-0.2 and 8.4+/-0.1. A plot of klim/Kd revealed a bell-shaped curve and two kinetically influential ionizations with macroscopic pKa values of 9.4+/-0.1 and 10.5+/-0.1. Mutagenesis of His-172, a potential active-site base and a component of a novel Tyr-His-Asp triad in the active site of TMADH, revealed that the pKa of 8.4+/-0.1 for the wild-type enzyme-substrate complex represents ionization of the imidazolium side-chain of His-172. H172Q TMADH retains catalytic competence throughout the pH range investigated. At pH 10.5, and in contrast with the wild-type enzyme, flavin reduction in H172Q TMADH is biphasic. The fast phase is dependent on the trimethylamine concentration and exhibits a kinetic isotope effect of about 3; C-H bond cleavage is thus partially rate-limiting. In contrast, the slow phase does not show hyperbolic dependence on substrate concentration, and the observed rate shows no dependence on isotope, revealing that C-H bond cleavage is not rate-limiting. The analysis of H172Q TMADH, together with data recently acquired for the Y169F mutant of TMADH, reveals that C-H bond breakage is not initiated via abstraction of a proton from the substrate by an active-site base. The transfer of reducing equivalents to flavin via a carbanion mechanism is therefore unlikely.
Department of Biochemistry, University of Leicester, Adrian Building, University Road, Leicester LE1 7RH, U.K.
J
J
Basran
M J
MJ
Sutcliffe
R
R
Hille
N S
NS
Scrutton
D004789:Enzyme Activation / Q000235:genetics; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology; D006639:Histidine; D007700:Kinetics; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D013379:Substrate Specificity / Q000235:genetics
D006639:Histidine; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase