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Kinetics of transhydrogenase reaction catalyzed by the mitochondrial NADH‐ubiquinone oxidoreductase (Complex I) imply more than one catalytic nucleotide‐binding sites
Author(s) -
Zakharova N.V.,
Zharova T.V.,
Vinogradov A.D.
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)00062-9
Subject(s) - submitochondrial particle , chemistry , ternary complex , oxidoreductase , kinetics , substrate (aquarium) , nucleotide , stereochemistry , catalysis , enzyme , redox , binding site , oxidase test , biochemistry , biology , organic chemistry , ecology , physics , quantum mechanics , gene
The steady‐state kinetics of the transhydrogenase reaction (the reduction of acetylpyridine adenine dinucleotide (APAD + ) by NADH, DD transhydrogenase) catalyzed by bovine heart submitochondrial particles (SMP), purified Complex I, and by the soluble three‐subunit NADH dehydrogenase (FP) were studied to assess a number of the Complex I‐associated nucleotide‐binding sites. Under the conditions where the proton‐pumping transhydrogenase (EC 1.6.1.1) was not operating, the DD transhydrogenase activities of SMP and Complex I exhibited complex kinetic pattern: the double reciprocal plots of the velocities were not linear when the substrate concentrations were varied in a wide range. No binary complex (ping‐pong) mechanism (as expected for a single substrate‐binding site enzyme) was operating within any range of the variable substrates. ADP‐ribose, a competitive inhibitor of NADH oxidase, was shown to compete more effectively with NADH ( K i =40 μM) than with APAD + ( K i =150 μM) in the transhydrogenase reaction. FMN redox cycling‐dependent, FP catalyzed DD transhydrogenase reaction was shown to proceed through a ternary complex mechanism. The results suggest that Complex I and the simplest catalytically competent fragment derived therefrom (FP) possess more than one nucleotide‐binding sites operating in the transhydrogenase reaction.