Open AccessKinetic and spectral studies on the redox forms of methanol dehydrogenase from Hyphomicrobium X
Author(s) -
FRANK Jzn Johannes,
DIJKSTRA Maarten,
DUINE Johannis A.,
BALNY Claude
Publication year - 1988
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1988.tb14102.x
Subject(s) - chemistry , methanol dehydrogenase , electron acceptor , redox , methanol , photochemistry , catalytic cycle , catalysis , substrate (aquarium) , half reaction , cofactor , enzyme , inorganic chemistry , organic chemistry , oceanography , geology
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, MDH red and MDH sem , 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 (MDH ox · s) into reduced enzyme (MDH red ) 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 MDH ox · C 2 H 3 OH complex could be isolated and its slow decomposition into MDH red 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].