[Coenzyme-induced slow transitions of NADP-sorbitol dehydrogenase from Gluconobacter oxydans].
The kinetic properties of NADP-dependent sorbitol dehydrogenase from G. oxydans cell extract were studied at pH 8.8 and 9.3 in the direction of D-sorbitol oxydation. It was shown that the shape of the kinetic curves of NADPH accumulation in time is characterised by initial burst whose magnitude depends on the concentration of the enzyme extract used. Preincubation of the enzyme with NADP or D-sorbitol eliminated the initial burst on these curves and transformed them into straight lines coming from the start of co-ordinates. The dependence of the stationary reaction rate on the enzyme extract concentration is not a linear one. The kinetic dependences of stationary rate of the reaction catalysed by the enzyme on the concentration of D-sorbitol and NADP at pH 8.8 and 9.3 were examined under all conditions studied; the shape of these kinetic curves altered to considerable extent with the alteration of the enzyme extract concentration in the reaction mixture and pH. At pH 9.3 several intermiediate plateaux were found on the curves of the D-sorbitol concentration dependent stationary rate of the reaction. The preincubation of the enzyme extract with NADP during 1.5 h removed the intermediate plateau on these curves and made them hyperbolic. Disk-electrophoresis of the enzyme extract in PAAG concentration gradient showed that at pH 8.8 the enzyme exists in one active form, while at pH 9.3 it exists in three major and three minor active forms of the enzyme differing in their molecular weights are found. It is assumed that the enzyme from G. oxydans cell extract can exist in a great number of molecular equilibrium forms, the rate of quilibrium being comparable or significantly less than that of the enzymatic reaction. NADP significantly influences on the equilibrium of the molecular forms of the enzyme.
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007064:L-Iditol 2-Dehydrogenase / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology*; D011548:Pseudomonadaceae / Q000201:enzymology*; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism*
D009249:NADP; D013401:Sugar Alcohol Dehydrogenases; D007064:L-Iditol 2-Dehydrogenase
Indutsiruemye kofermentom medlennye perekhody NADP-sorbitoldegidrogenazy iz Gluconobacter oxydans.