Unno M, Ishimori K, Ishimura Y, Morishima I
Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
Biochemistry. 1994 Aug 16;33(32):9762-8. doi: 10.1021/bi00198a047.
The effects of camphor and camphor analogues on the CO recombination kinetics of ferrous cytochrome P450CAM (P450CAM) at 293 K have been studied as a function of hydrostatic pressure (0.1-200 MPa) by means of flash photolysis. At 0.1 MPa, the association rate constant (kon) for substrate-free P450CAM is 8.5 x 10(6) M-1 s-1. Measurements as a function of pressure lead to a determination of the activation volume (delta V not equal to) of +4 cm3 mol-1 for substrate-free protein. This positive delta V not equal to is interesting because the CO association reaction of various hemoproteins, such as myoglobin and hemoglobin, exhibit negative delta V not equal to values [Adachi, S., & Morishima, I. (1989) J. Biol. Chem. 264, 18896-18901; Unno, M., Ishimori, K., & Morishima, I. (1990) Biochemistry 29, 10199-10205]. The binding of d-camphor and some camphor analogues (d-fenchone, 3-endo-bromocamphor, and 3,3,5,5-tetramethylcyclohexanone) into the heme pocket strongly influences the kinetics, i.e., kon is reduced ((1-10) x 10(5) M-1 s-1) and delta V not equal to is altered to a negative value (-14 to -32 cm3 mol-1). The negative delta V not equal to suggests that the effects of camphor and these camphor analogues are due to an increase in the iron-ligand bond formation barrier. On the other hand, the binding of adamantane and norcamphor does not affect the kinetics. This result is particularly surprising because both substrate analogues are located in the immediate vicinity of the CO binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
在293K下，通过闪光光解研究了樟脑及其类似物对亚铁细胞色素P450CAM（P450CAM）的CO重组动力学的影响，并将其作为静水压力（0.1 - 200MPa）的函数。在0.1MPa下，无底物P450CAM的缔合速率常数（kon）为8.5×10⁶ M⁻¹ s⁻¹。作为压力函数的测量结果确定了无底物蛋白质的活化体积（ΔV≠）为 +4 cm³ mol⁻¹。这个正的ΔV≠很有趣，因为各种血红素蛋白（如肌红蛋白和血红蛋白）的CO缔合反应表现出负的ΔV≠值[安达，S.，& 森岛，I.（1989）《生物化学杂志》264，18896 - 18901；宇野，M.，石森，K.，& 森岛，I.（1990）《生物化学》29，10199 - 10205]。d - 樟脑和一些樟脑类似物（d - 葑酮、3 - 内 - 溴樟脑和3,3,5,5 - 四甲基环己酮）结合到血红素口袋中会强烈影响动力学，即kon降低（（1 - 10）×10⁵ M⁻¹ s⁻¹），并且ΔV≠变为负值（-14至 -32 cm³ mol⁻¹）。负的ΔV≠表明樟脑和这些樟脑类似物的影响是由于铁 - 配体键形成障碍的增加。另一方面，金刚烷和降樟脑的结合不影响动力学。这个结果特别令人惊讶，因为这两种底物类似物都位于CO结合位点的紧邻区域。（摘要截断于250字）
