Tordi M G, Silvestrini M C, Colosimo A, Tuttobello L, Brunori M
Biochem J. 1985 Sep 15;230(3):797-805. doi: 10.1042/bj2300797.
The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.
对铜绿假单胞菌细胞色素氧化酶催化的天青蛋白和细胞色素c-551的氧化动力学进行了重新研究，并通过参数法和非参数法评估了稳态参数。在低底物浓度下（例如小于或等于50微摩尔），天青蛋白的米氏常数（Km）和催化中心活性的值分别为15±3微摩尔和77±6分钟⁻¹，细胞色素c-551的分别为2.15±0.23微摩尔和66±2分钟⁻¹，总体上与先前报道一致，即细胞色素c-551对该酶具有更高的亲和力，而天青蛋白具有略高的催化速率。然而，当细胞色素c-551的浓度大大超过Km值时，初始速度增加，最终在底物浓度大于或等于100微摩尔时几乎翻倍。这一结果表明存在一种“半心半意”的行为，因为在相对较低的细胞色素c-551浓度下，二聚体酶的两个相同结合位点中似乎只有一个具有催化活性，这可能是由于不利的相互作用影响了第二个位点上米氏复合物的稳定性。当还原型天青蛋白和细胞色素c-551同时暴露于铜绿假单胞菌细胞色素氧化酶时，观察到的稳态氧化动力学很复杂，鉴于细胞色素c-551和天青蛋白在游离状态下能快速进行电子转移，这是预期的。尽管存在这种复杂性，但似乎一种涉及两种底物在酶的同一活性位点上简单竞争的机制在起作用。添加化学修饰且无氧化还原活性的天青蛋白形式（汞-天青蛋白）对天青蛋白和细胞色素c-551的初始氧化速率均无影响，但通过至少部分消除产物抑制作用，明显改变了整个过程的时间进程。结果得出以下结论：（i）还原型天青蛋白和细胞色素c-551在酶的同一位点结合，因此存在竞争；（ii）汞-天青蛋白在一个调节位点结合，与产物而非底物竞争；（iii）二聚体酶上的两个结合位点虽然本质上是等效的，但存在不利的相互作用。由于水是氧气还原的产物，（iii）点对反应机制具有重要意义。
