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Schulze,H., G. Hui Bon Hoa, V. Helms, R.C. Wade and C. Jung (1996)
Structural Changes in Cytochrome P-450cam Effected
by the Binding of
the Enantiomers (1R)-Camphor and (1S)-Camphor.
submitted to
Biochemistry.
A comparative study of the enantiomeric substrate- ((1R)-camphor- and (1S)- camphor-) bound cytochrome P-450cam concerns the spin equilibrium, substrate dissociation, thermal unfolding of the protein structure, and the subconformer equilibria observed in the infrared spectra of the carbonmonoxide (CO) complex of cytochrome P-450cam. The behaviour of the different conformational equilibria in dependence on temperature, pressure, pH-value, co-solvent and cation binding led us to suggest that (1S)-camphor is more loosely and less optimal bound in the heme pocket, which facilitates the access of solvent molecules into the heme-iron environment. The spin reaction volume difference measured using the high pressure technique is smaller by 16 +/- 9 cm3/mol for (1S)-camphor-bound P- 450cam compared to the (1R)-camphor-bound, which might indicate a higher water content in the protein and in the heme environment in the (1S)-camphor complex. The half transition temperature of the thermal unfolding of 53.8oC for the (1S)-camphor-bound oxidised cytochrome P-450cam is 1 degree lower than the value for the (1R)-camphor-bound protein (54.8oC). in the reduced, CO-bound form of the cytochrome P-450cam at 290K the (1S)-camphor complex reveals another CO stretch vibration population distribution with slightly higher frequencies (1940.2 cm-1) (major band) and 1946.3 cm-1 (minor band) compared to the (1R)-camphor complex (1939.7 cm-1) (major band) and 1930 (minor band)). A loosening of the contact between the iron-bound CO ligand and amino acids of the I-helix, probably induced by compensating effects of the inceased water content, is suggested. Assuming the carbonmonoxide complex as a model for the dioxygen complex, the more loosened binding of (1S)-camphor, therefore the increased water accessibility, and the weaker contact of the CO ligand to the I-helix might explain the higher amount of uncoupling of the cytochrome P-450 reaction cycle from insertion of the oxygen atom into (1S)-camphor compared to that of (1R)-camphor.
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Wade et al. (1996) Fundamentals of Enzyme-Ligand Interactions in
Cytochrome P450cam
POPE5 Conference Proceedings
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