Single turnover kinetics of the reaction between oxycytochrome P-450cam and reduced putidaredoxin.
A study of the single turnover kinetics of the reaction between oxycytochrome P-450cam and reduced putidaredoxin was performed using the inhibitor metyrapone to trap the cytochrome immediately after release of the product, 5-exo-hydroxycamphor. EPR determinations of the concentrations of reduced putidaredoxin and ferric metyrapone-bound cytochrome at the same time points showed that there is no time lag between the oxidation of reduced putidaredoxin and the appearance of metyrapone-bound cytochrome. This implies that the rate constant for electron transfer is smaller than the rate constant for the later processes involved in product formation and release, lumped into a single step. Taking this restriction into account and doing computer simulation of absorbance versus time curves, previously obtained at various putidaredoxin concentrations using stopped-flow spectrophotometry, allowed bounds to be determined for rate constants of the processes within the reaction. At 4 degrees C in buffer at pH 7.4 with 0.50 M KCl, the rate constant for the bimolecular association of the two enzymes is between 3 and 20/microM.s; the rate constant for dissociation is between 12 and 600/s; the rate constant for electron transfer is between 60 and 100/s; and the rate constant for the later processes is at least 200/s.
Brewer
C B
CB
Department of Biochemistry, University of Texas Health Science Center at Dallas 75235.
Peterson
J A
JA
D019475:Camphor 5-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005288:Ferredoxins / Q000378:metabolism*; D007700:Kinetics; D008797:Metyrapone / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology
D005288:Ferredoxins; C100137:putidaredoxin; D011189:Potassium Chloride; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase; D008797:Metyrapone