Mobility of norbornane-type substrates and water accessibility in cytochrome P-450cam.
The behaviour of norbornane-type substrates bound to oxidised cytochrome P-450cam (CYP 101) in 60% (w/w) glycerol-containing phosphate buffer was investigated using electronic absorption spectroscopy. The high-pressure dependence study revealed that the value of the spin-state reaction-volume change decreased from -70 to -22.8 cm3/mol with decreasing high-spin state content from 99 to 63%. Simultaneously, the values for the enthalpy and entropy determined from the low-temperature dependence of the spin-state transition decreased from 73.7 to 24.3 kJ/mol and from 310.4 to 88.9 J/mol K, respectively. Under our experimental conditions the pH-value of the buffer remained at low temperatures and high pressures in the range of pH 7-8, in which no pH-value-induced spin-state conversion occurred. Therefore, the secondary effect of the temperature and pressure-induced pH change can be disregarded as being responsible for the observed spin-state transition effects. Substrate dissociation constants were determined. From the temperature-jump experiments (297 K to 180 K) we found a higher mobility in the active site for the substrates in the sequence (1R)-camphor, (1S)-camphor, camphane, (1R)- and (1S)-camphorquinone, norcamphor, and norbornane. Our findings can be explained by the incomplete fit of the methyl groups of the norbornane-type substrate to the protein, in particular to the I-helix, predominantly determining the substrate mobility and water accessibility to the protein.
Schulze
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Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany.
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Jung
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D001665:Binding Sites; D002151:Calorimetry; D002164:Camphor / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015394:Molecular Structure; D009636:Norbornanes / Q000737:chemistry* / Q000378:metabolism; D011312:Pressure; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D009636:Norbornanes; D011994:Recombinant Proteins; D002164:Camphor; D019475:Camphor 5-Monooxygenase