Mechanism of yeast cytochrome b2 action. I. Thermodynamics and relaxation kinetics of the interaction between cytochrome b2 and oxalate.
Oxalate is the strongest known inhibitor of yeast cytochrome b2 activity. We have used spectrophotometric titration, temperature-jump relaxation, and calorimetry in an investigation of the interaction between enzyme and inhibitor. The titration data are consistent with noncooperative binding to one site per subunit. This conclusion is corroborated by temperature-jump results which reveal a single relaxation phenomenon which obeys second-order kinetics. Further evidence for a simple binding reaction enthalpy estimated from relaxation amplitudes is in good agreement with the value obtained directly with batch calorimetry. The forward and reverse rate constants evaluated from the temperature-jump experiments are, respectively, 1 x 10(4) M-1 sec-1 and 15 sec-1. Although considerably smaller than a diffusion-controlled value, the forward rate constant is characterized by an unusually small activation energy of approximately 3 kcal/mol. This, together with a large unfavorable association activation entropy of -30 eu, suggests that oxalate diffuses freely to the active site, but only a small fraction of the collisions are productive due to severe steric requirements.
D001665:Binding Sites; D003580:Cytochromes / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D010070:Oxalates; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013053:Spectrophotometry; D013696:Temperature
D003580:Cytochromes; D010070:Oxalates