Kinetic studies on the diphosphoryridine nucleotide cytochrome c reductase from heart

1. 1. Initial velocities for the reaction catalyzed by DPNH-cytochrome c reductase from pig heart sarcosomes have been determined at 14° as a function of DPNH, cytochrome c and hydrogen ion concentration. 2. 2. Maximum velocities and Michaelis constants have been calculated from the data over a pH range of 7 to 9.2. 3. 3. At constant pH, the kinetic results follow the rate equation ν = V I+ K_DPNH [DPNH]1+ K_cyt [cyt]which indicates that the enzymic reaction may be treated as a "two substrate" case even though the overall stoichiometry of the reaction requires three substrate molecules. 4. 4. The pH-dependence of the maximum velovity indicates that three ionizing groups are involved in the enzymic reaction. Furthermore, the pH-dependence of the maximum velocity-Michaelis constant ratio shows that two of the three groups are in the enzymically active site and are associated with the oxidation of DPNH. The third ionizing group is involved in the reduction of cytochrome c. It is impossible to determine kinetically whether this third grouo is associated with the enzyme or with the cytochrome itself. 5. 5. Ionization constants for the groups involved and the so-called pH-indepedent kinetic parameters have been calculated. 6. 6. The β-deuterium labelled reduced DPN has been made by the stereospecific exchange reaction catalyzed by the enzyme. Use of this material as a substrate shows that the Michaelis constant for DPNH in unaffected but that the maximum velocity is decreased approximately 2.3 fold.