Open AccessInvestigations on the Kinetic Mechanism of Octopine Dehydrogenase
Author(s) -
DOUBLET MarieOdile,
OLOMUCKI Anna,
BAICI Antonio,
LUISI Pier Luigi
Publication year - 1975
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1975.tb02440.x
Subject(s) - octopine , kinetic isotope effect , chemistry , cofactor , dehydrogenase , substrate (aquarium) , enzyme , enzyme kinetics , nad+ kinase , deuterium , stereochemistry , biochemistry , active site , biology , transformation (genetics) , physics , ecology , quantum mechanics , gene , agrobacterium
The kinetic mechanism of octopine dehydrogenase has been investigated by stopped‐flow and isotope replacement techniques. When the enzyme is saturated by substrate and coenzyme, both for NADH oxidation and NAD + reduction, the stationary phase is preceded by a rapid burst. Under these saturation conditions, furthermore, the stationary phase shows a secondary isotope effect when 4 S ‐[4‐ 2 H]NADH is substituted for NADH and when (on the other reaction end) d ‐[ 2 H]octopine is substituted for d ‐octopine. The data are taken to indicate that the rate‐limiting step for enzyme turnover is a step following a very fast chemical transformation of the reagents. However, when the substrate concentration is lowered below the corresponding K m value, keeping the coenzyme concentration at saturating levels, the time course of the reaction shows no burst and the stationary phase has a larger isotope effect. This indicates that under those non‐saturating conditions, the enzyme turnover has a larger contribution than the hydrogen‐transfer step. Changing the coenzyme concentration alone has very little or no effect on the amplitude of the burst or on the isotope effect. These features are discussed in terms of the other known kinetic properties of the enzyme, and in terms of analogous studies reported in the literature for other dehydrogenases.