Studies on the reaction mechanism of DT diaphorase

Studies on high molecular weight modifiers of the nonhyperbolic V versus [S] curves of DT‐diaphorase

“…The effect of pH and temperature on the V vs [S] plots of DT-diaphorase has been shown previously [4]. The use of these nonspecific effectors of protein conformation resulted in changes in the V vs [S] curves.…”

“…wt over 60 000 may be an interaction between the enzyme and the effector leading to the stabilization of one of the two enzyme species present. It was suggested in a previous report [4] that the basis for the complex V vs [S] curves is that two enzyme species in slow isomerization equilibrium with each other are present.…”

“…DT-diaphorase was prepared from the soluble fraction of rat liver homogenates, essentially by the method previously described by Ernster et al [1] and modified as described [4]. The assay medium, in a volume of 1 ml consisted of 50 mM Tris-chloride pH 7.5, NADP and DCPIP and modifier.…”

“…Previous studies have shown that the V vs IS] curves of this enzyme are not hyperbolic: The Vvs [NADH] curve has an intermediary plateau region at about 0.3 mM substrate [2,3] and the V vs [DCPIP] plot has a 'trough' (inverted peak) region between approx. 25-37/~M of this acceptor [4]. The complex kinetic curves have been interpreted as reflecting slow isomerization between two enzyme species each having two sites or possibly negative and positive cooperativity between subunits or sites of a multisite enzyme [4].…”

“…The complex kinetic curves have been interpreted as reflecting slow isomerization between two enzyme species each having two sites or possibly negative and positive cooperativity between subunits or sites of a multisite enzyme [4]. Temperature, pH, salts [4], ethanol and sucrose Hollander, unpublished results) affected both the Vvs [NADH] and the Vvs [DCPIP] curves. Previous experiments showed that nonionic detergents (Tween 20) [1] and phospholipids [5] increased the enzyme dependent reaction rates.…”