Coenzyme binding capacity of yeast alcohol dehydrogenase.

Commercial lyophilized preparations of yeast alcohol dehydrogenase from Boehringer G.m.b.H. (Mannheim, Germany) bind 2 mols of reduced coenzyme/144000 g of enzyme (1). After the purification by a DEAE-Sephadex column chromatography, the coenzyme binding capacity is raised to 4 mols of NADH/mol of enzyme. Commercial preparations and ionexchange-purified preparations are homogeneous on the ionexchange column chromatography and the disc gel electrophoresis, after reduction with thioglycolic acid. Ionexchange chromatography does not increase the -SH titer, zinc content and the specific activity of enzyme. It is suggested that ionexchange chromatography raises the NADH-binding capacity by removing some impurities present in commercial enzyme preparations.     

Coenzyme Q: a molecular orbital study.

The molecular structure and electronic properties of coenzyme Q, also known as ubiquinone, have been studied with the methods of semiempirical molecular orbital theory. It is found that the preferred conformation of the isoprene unit is such that formation of the chroman is favorable. The methoxy groups then assume a nonplanar configuration. Orbital energies, dipole moments, and spectra for the coenzyme Q₁ molecule and some of its chroman intermediates are also given in the calculations. The possibility of charge-transfer complex formation of chroman intermediate with inorganic phosphate is discussed.     

Coenzyme Q10 treatment in serious heart failure.

Several noninvasive studies have shown the effect on heart failure of treatment with coenzyme Q10. In order to confirm this by invasive methods we studied 22 patients with mean left ventricular (LV) ejection fraction 26%, mean LV internal diameter 71 mm and in NYHA class 2-3. The patients received coenzyme Q10 100 mg twice daily or placebo for 12 weeks in a randomized double-blinded placebo controlled investigation. Before and after the treatment period, a right heart catheterisation was done including a 3 minute exercise test. The stroke index at rest and work improved significantly, the pulmonary artery pressure at rest and work decreased (significantly at rest), and the pulmonary capillary wedge pressure at rest and work decreased (significantly at 1 min work). These results suggest improvement in LV performance. Patients with congestive heart failure may thus benefit from adjunctive treatment with coenzyme Q10.     

Coenzyme dependent suppression of 2,2-PDS induced contractures in Spirostomum.

Spirostomum ambiguum was stimulated to contract by addition of the thiol inhibitor, 2,2-dithiobispyridine (PDS), which depletes intracellular reducing equivalents which are generated by coenzyme dependents reactions. PDS induced contractures were not suppressed after 2--4 h of pre-incubation in a medium enriched with ascorbic acid, thiamine, or riboflavin, singly. Two h of incubation with nicotinamide did not suppress contractures; however, 50% suppression occurred after 3 h incubation. Incubation of the cells in a medium enriched with all four coenzymes for up to 4 h, resulted in the suppression of PDS induced contractures to a level as low as 30% of control values. Suppression of contractures by the mixed coenzymes was concentration dependent. Cells that were stimulated with PDS and contracting, exhibited a 50% suppression of contractures within 3 min after transfer to a complementary medium enriched with mixed coenzymes. These results suggest that coenzymes interact synergistically with cellular metabolic processes to inhibit pharmacodynamic responses to PDS.     

Synthesis of S-Behenyl Coenzyme A

The synthesis of behenyl CoA by the reaction of coenzyme A with N-hydroxysuecinimide ester of behenic acid is reported. This method gives better yields of behenyl CoA when compared to the acid chloride or mixed anhydride method where side reactions seem to cause a decrease in yield.     

Coenzyme Q10, a cutaneous antioxidant and energizer.

The processes of aging and photoaging are associated with an increase in cellular oxidation. This may be in part due to a decline in the levels of the endogenous cellular antioxidant coenzyme Q10 (ubiquinone, CoQ10). Therefore, we have investigated whether topical application of CoQ10 has the beneficial effect of preventing photoaging. We were able to demonstrate that CoQ10 penetrated into the viable layers of the epidermis and reduce the level of oxidation measured by weak photon emission. Furthermore, a reduction in wrinkle depth following CoQ10 application was also shown. CoQ10 was determined to be effective against UVA mediated oxidative stress in human keratinocytes in terms of thiol depletion, activation of specific phosphotyrosine kinases and prevention of oxidative DNA damage. CoQ10 was also able to significantly suppress the expression of collagenase in human dermal fibroblasts following UVA irradiation. These results indicate that CoQ10 has the efficacy to prevent many of the detrimental effects of photoaging.     

Coenzyme A- and NADH-dependent esterase activity of methylmalonate semialdehyde dehydrogenase.

Methylmalonate semialdehyde dehydrogenase purified to homogeneity from rat liver possesses, in addition to its coupled aldehyde dehydrogenase and CoA ester synthetic activity, the ability to hydrolyze p-nitrophenyl acetate. The following observations suggest that this activity is an active site phenomenon: (a) p-nitrophenyl acetate hydrolysis was inhibited by malonate semialdehyde, substrate for the dehydrogenase reaction; (b) p-nitrophenyl acetate was a strong competitive inhibitor of the dehydrogenase activity; (c) NAD+ and NADH activated the esterase activity; (d) coenzyme A, acceptor of acyl groups in the dehydrogenase reaction, accelerated the esterase activity; and (e) the product of the esterase reaction proceeding in the presence of coenzyme A was acetyl-CoA. These findings suggest that an S-acyl enzyme (thioester intermediate) is likely common to both the esterase reaction and the aldehyde dehydrogenase/CoA ester synthetic reaction.     

Propionyl-CoA condensing enzyme from Ascaris muscle mitochondria. II. Coenzyme A modulation.

The propionyl-CoA condensing enzyme which catalyzes the first step in the biosynthesis of 2-methylbutyrate and 2-methylvalerate by Ascaris muscle appears to exist in at least three forms in the mitochondria of this parasitic nematode. Two forms, A and B, were separated by ion exchange chromatography on CM-Sephadex. Chromatography on phosphocellulose resulted in the recovery of one minor peak (I) and two major peaks with activity (II and III). A and B as well as I, II, and III differed in their specific activities. Forms B and III were the most retained by their resins, and were the most active forms of the enzyme in each case. Inhibition studies with metabolites from Ascaris mitochondria indicate that CoASH, a product of the condensation reaction, and acetyl-CoA are effective inhibitors of the condensing and thiolytic activities of the Ascaris enzyme, respectively. Incubation of the active enzyme form B for 2 h with 0.1 mM CoASH at room temperature under nitrogen caused the loss of 92% of the propionyl-CoA condensing activity and 67% of the thiolase activity when assayed in standard mixtures. The propionyl-CoA condensing enzyme exhibited a hyperbolic dependence of the condensation velocity to changes in substrate concentration. However, in the presence of CoASH the Michaelis-Menten kinetics was transformed into a sigmoidal kinetics indicating a deviation from a simple product inhibition. Inactivation of the most active forms of the enzyme with CoASH was accompanied by (a) a change in the chemical reactivity of the protein toward p-chloromurcuribenzoate, (b) a change in the uv-visible spectrum of the protein, and (c) a change in the elution patterns from both CM-Sephadex and phosphocellulose column chromatography, where-upon one, two, or more protein peaks were obtained. The several protein peaks resolved by rechromatography of the [14C]CoASH-inactivated enzyme III on phosphocellulose had different CoASH contents. The elution positions were correlated with the less active forms (I and II) having increased [14C]CoASH activities. Similarly, the two peaks isolated upon rechromatography of the CoASH-partially inactivated enzyme B on CM-Sephadex had different isotope contents and the elution position of enzyme A corresponded to the less active form. The results described indicate that the CoASH modification of Ascaris propionyl-CoA condensing enzyme may be responsible for the existence of several forms of the enzyme and might represent a mode of control by chemically modulating the amount of the active forms of the enzyme.     

Coenzyme specificity in aldehyde dehydrogenase

Influences on coenzyme preference are explored. Lysine 137 (192 in class 1/2 ALDH) lies close to the adenine ribose, directly interacting with the adenine ribose in NAD-specific ALDHs and the 2'-phosphate of NADP in NADP-specific ALDHs. Lys-137 in class 3 ALDH interacts with the adenine ribose indirectly through an intervening water molecule. However, this residue is present in all ALDHs and, as a result, is unlikely to directly influence coenzyme specificity. Glutamate 140 (195) coordinates the 2'- and 3'-hydroxyls of the adenine ribose of NAD in the class 3 tertiary structure. Thus, it appeared that this residue would influence coenzyme specificity. Mutation to aspartate, asparagine, glutamine or threonine shifts the coenzyme specificity towards NADP, but did not completely change the specificity. Still, the mutants show the 2'-phosphate of NADP is repelled by Glu-140 (195). Although Glu-140 (195) has a major influence on coenzyme specificity, it is not the only influence since class 3 ALDHs, can use both coenzymes, and class 2 ALDHs, which are NAD-specific, have a glutamate at this position. One explanation may be that the larger space between Lys-137 (192) and the adenine ribose hydroxyls in the class 3 ALDH:NAD binary structure may provide space to accommodate the 2'-phosphate of NADP. Also, a structural shift upon binding NADP may also occur in class 3 ALDHs to help accommodate the 2'-phosphate of NADP.