Schulze H, Hoa G H, Jung C
Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany.
Biochim Biophys Acta. 1997 Mar 7;1338(1):77-92. doi: 10.1016/s0167-4838(96)00192-6.
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.
利用电子吸收光谱法研究了在含60%（w/w）甘油的磷酸盐缓冲液中，与氧化型细胞色素P-450cam（CYP 101）结合的降冰片烷型底物的行为。高压依赖性研究表明，随着高自旋态含量从99%降至63%，自旋态反应体积变化值从-70降至-22.8 cm³/mol。同时，由自旋态转变的低温依赖性确定的焓值和熵值分别从73.7降至24.3 kJ/mol和从310.4降至88.9 J/mol K。在我们的实验条件下，缓冲液的pH值在低温和高压下保持在pH 7-8范围内，在此范围内不会发生pH值诱导的自旋态转变。因此，温度和压力诱导的pH变化的二次效应可被视为与观察到的自旋态转变效应无关而不予考虑。测定了底物解离常数。通过温度跳跃实验（从297 K到180 K），我们发现底物在活性位点的迁移率按以下顺序升高：（1R）-樟脑、（1S）-樟脑、莰烷、（1R）-和（1S）-樟脑醌、降樟脑和降冰片烷。我们的发现可以用降冰片烷型底物的甲基与蛋白质，特别是与I-螺旋不完全匹配来解释，这主要决定了底物的迁移率和蛋白质的水可及性。
