Single turnover studies with oxy-cytochrome P-450cam

Archives of Biochemistry and Biophysics
C B Brewer, J A Peterson

Abstract

The catalytic step of bacterial cytochrome P-450cam, i.e., the step of the reaction cycle in which the product 5-exo-hydroxycamphor is formed and released by the enzyme, has been studied by stopped-flow spectrophotometry. Our approach has been to observe a single-turnover reaction between reduced putidaredoxin and oxygenated camphor-bound cytochrome P-450cam. Multiple turnovers are prevented by using the inhibitor metyrapone to trap the cytochrome after product release, which prevents binding of another camphor molecule. The time course of the reaction has been measured at several wavelengths and has been found to be biphasic. The relatively slow second phase of the reaction is the reduction of ferric, metyrapone-bound cytochrome P-450cam. The first phase coincides with the formation of product stoichiometrically with cytochrome P-450cam, as measured by gas chromatography. A detailed kinetic study of the first phase reveals a hyperbolic dependence of initial rate upon putidaredoxin concentration at a fixed, limiting concentration of cytochrome P-450cam. The Vmax is 53 microM per second per microM cytochrome, and the Km for putidaredoxin is 33 microM. The hyperbolic relationship between initial rate and putidaredoxin concentrati...Continue Reading

References

Oct 1, 1975·Analytical Biochemistry·J A Peterson, D M Mock
Aug 1, 1971·Archives of Biochemistry and Biophysics·J A PetersonA G Hildebrandt

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Citations

Dec 8, 2005·Applied and Environmental Microbiology·Effendi LeonardMattheos A G Koffas
Jan 10, 2014·Chemical Reviews·Thomas L Poulos
Feb 12, 1998·Proceedings of the National Academy of Sciences of the United States of America·V ReipaV L Vilker
Mar 3, 2006·Journal of Inorganic Biochemistry·Thomas M MakrisStephen G Sligar
Jun 9, 2005·Chemical Reviews·Ilia G DenisovIlme Schlichting
Aug 9, 2005·Biochemical and Biophysical Research Communications·Swati PrasadSamaresh Mitra
May 16, 2002·Journal of the American Chemical Society·Laura S KooPaul R Ortiz de Montellano
Mar 4, 2000·Science·I SchlichtingS G Sligar

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