Kinetic and spectral studies on the redox forms of methanol dehydrogenase from Hyphomicrobium X.
Several reaction rate constants in the catalytic cycle of methanol dehydrogenase (EC 1.1.99.8) in vitro were determined with stopped-flow spectrophotometry. The studies revealed that the high pH required for adequate activity of the enzyme is related to the strong pH dependency of the oxidation rates of the reduced and semiquinone enzyme forms, MDHred and MDHsem, with the artificial electron acceptor Wurster's blue. The rate-limiting step in the catalytic cycle is associated with the conversion of oxidized enzyme-substrate complex (MDHox.S) into reduced enzyme (MDHred) and product. The effect of activator (ammonium salts) was also confined to this step, but even saturating concentrations were unable to remove the limitation completely. Making use of the large deuterium isotope effect associated with substrate oxidation in the absence of activator, the transient MDHox.C2H3OH complex could be isolated and its slow decomposition into MDHred and formaldehyde could be demonstrated. Further evidence is presented to support the view that the different absorption spectra observed originate from genuine redox forms of methanol dehydrogenase with different redox states of the cofactor PQQ and not from enzyme-electron acceptor complexes. The results confirm and extend our original view on the mechanism of action and contradict the mechanism proposed by others [Parkes, C. & Abeles, R. H. (1984) Biochemistry 23, 6355-6363].
Frank
J
J
Laboratory of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
Dijkstra
M
M
Duine
J A
JA
Balny
C
C
D000429:Alcohol Oxidoreductases / Q000032:analysis*; D001419:Bacteria / Q000201:enzymology*; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor)