Kinetic characterization of the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae.
Saccharomyces cerevisiae mitochondria contain an NADH:Q6 oxidoreductase (internal NADH dehydrogenase) encoded by NDI1 gene in chromosome XIII. This enzyme catalyzes the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. From a structural point of view, the mature enzyme has a single subunit of 53 kDa with FAD as the only prosthetic group. Due to the fact that S. cerevisiae cells lack complex I, the expression of this protein is essential for cell growth under respiratory conditions. The results reported in this work show that the internal NADH dehydrogenase follows a ping-pong mechanism, with a Km for NADH of 9.4 microM and a Km for oxidized 2,6-dichorophenolindophenol (DCPIP) of 6.2 microM. NAD+, one of the products of the reaction, did not inhibit the enzyme while the other product, reduced DCPIP, inhibited the enzyme with a Ki of 11.5 microM. Two dead-end inhibitors, AMP and flavone, were used to further characterize the kinetic mechanism of the enzyme. AMP was a linear competitive inhibitor of NADH (Ki = 5.5 mM) and a linear uncompetitive inhibitor of oxidized DCPIP (Ki = 11.5 mM), in agreement with the ping-pong mechanism. On the other hand, flavone was a partial inhibitor displaying a hyperbolic uncompetitive inhibition regarding NADH, and a hyperbolic noncompetitive inhibition with respect to oxidized DCPIP. The apparent intercept inhibition constant (Kii = 5.4 microM) and the slope inhibition constant (Kis = 7.1 microM) were obtained by non linear regression analysis. The results indicate that the ternary complex F-DCPIPox-flavone catalyzes the reduction of DCPIP, although with lower efficiency. The effect of pH on Vmax was studied. The Vmax profile shows two groups with pKa values of 5.3 and 7.2 involved in the catalytic process.
Velázquez
I
I
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, DF, México.
Pardo
J P
JP
D015086:2,6-Dichloroindophenol / Q000378:metabolism / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D042967:Electron Transport Complex I; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D047309:Flavones; D005419:Flavonoids / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000201:enzymology*; D009243:NAD / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D011809:Quinones / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D047309:Flavones; D005419:Flavonoids; D011809:Quinones; D012402:Rotenone; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate; D015086:2,6-Dichloroindophenol; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; C043562:flavone