Imai M, Shimada H, Watanabe Y, Matsushima-Hibiya Y, Makino R, Koga H, Horiuchi T, Ishimura Y
Department of Biochemistry, School of Medicine, Keio University, Tokyo, Japan.
Proc Natl Acad Sci U S A. 1989 Oct;86(20):7823-7. doi: 10.1073/pnas.86.20.7823.
Site-directed mutants of cytochrome P-450cam (the cytochrome P-450 that acts as the terminal monooxygenase in the d-camphor monooxygenase system), in which threonine-252 had been changed to alanine, valine, or serine, were employed to study the role of the hydroxy amino acid in the monooxygenase reaction. The mutant enzymes were expressed in Escherichia coli and were purified by a conventional method. All the mutant enzymes in the presence of d-camphor exhibited optical absorption spectra almost indistinguishable from those of the wild-type enzyme in their ferric, ferrous, oxygenated, and carbon monoxide ferrous forms. In a reconstituted system with putidaredoxin and its reductase, the alanine enzyme consumed O2 at a rate (1100 per min per heme) comparable to that of the wild-type enzyme (1330 per min per heme), whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type enzyme. About 85% of the O2 consumed was recovered as H2O2. The valine enzyme also exhibited an oxidase activity to yield H2O2 accompanied by a relative decrease in the monooxygenase activity. On the other hand, the serine enzyme exhibited essentially the same monooxygenase activity as that of the wild-type enzyme. Thus, uncoupling of O2 consumption from the monooxygenase function was produced by the substitution of an amino acid without a hydroxyl group. When binding of O2 to the ferrous forms was examined, the alanine and valine enzymes formed instantaneously an oxygenated form, which slowly decomposed to the ferric form with rates of 5.5 and 3.2 x 10(-3) sec-1 for the former and latter enzymes, respectively. Since these rates were too slow to account for the overall rates of O2 consumption, the formation of H2O2 was considered to proceed not by way of this route but through the decomposition of a peroxide complex formed by reduction of the oxygenated form by reduced putidaredoxin. Based on these findings, a possible mechanism for oxygen activation in this monooxygenase reaction has been discussed.
细胞色素P-450cam（在d-樟脑单加氧酶系统中作为末端单加氧酶的细胞色素P-450）的定点突变体，其中苏氨酸-252已被替换为丙氨酸、缬氨酸或丝氨酸，用于研究羟基氨基酸在单加氧酶反应中的作用。突变酶在大肠杆菌中表达，并通过常规方法纯化。在d-樟脑存在下，所有突变酶在其铁、亚铁、氧合和一氧化碳亚铁形式下的光吸收光谱与野生型酶几乎无法区分。在与恶臭假单胞菌铁氧还蛋白及其还原酶重构的系统中，丙氨酸酶消耗O2的速率（每血红素每分钟1100）与野生型酶（每血红素每分钟1330）相当，而形成的外-5-羟基樟脑量不到野生型酶形成量的10%。消耗的O2约85%以H2O2形式回收。缬氨酸酶也表现出氧化酶活性，产生H2O2，同时单加氧酶活性相对降低。另一方面，丝氨酸酶表现出与野生型酶基本相同的单加氧酶活性。因此，通过替换无羟基的氨基酸导致了O2消耗与单加氧酶功能的解偶联。当检测O2与亚铁形式的结合时，丙氨酸和缬氨酸酶立即形成氧合形式，前者和后者的氧合形式分别以5.5和3.2×10(-3)秒-1的速率缓慢分解为铁形式。由于这些速率太慢，无法解释O2消耗的总体速率，因此认为H2O2的形成不是通过这条途径，而是通过还原型恶臭假单胞菌铁氧还蛋白还原氧合形式形成的过氧化物复合物的分解。基于这些发现，讨论了该单加氧酶反应中氧激活的可能机制。
