Kuma F
J Biol Chem. 1981 Jun 10;256(11):5518-23.
A soluble erythrocyte cytochrome b5 was purified as the substrate of methemoglobin reductase and an electron carrier to methemoglobin. The isoelectric point of this protein was at pH 4.3, and E0' was -0.010 at pH 7.0.. The Km value of the enzyme for this protein was 1 x 10(-4) M, and the turnover number (k5) was 3.4 x 10(4) min-1, with NADH as an electron donor at pH 7.0. The optimum pH of the enzyme was pH 4.6 for ferricyanide and pH 5.5 for cytochrome b5, with a shoulder of activity at pH 7 to 9 for both substrates. The rate equation which represents the reduction of either methemoglobin or cytochrome c was obtained as a function of methemoglobin or cytochrome c, methemoglobin reductase, and cytochrome b5 by considering the E . S complex for both reductase and cytochrome b5, and the rate constants involved were determined. The rate constants between methemoglobin and reduced cytochrome b5 (k1, M-1 min-1) were 1.6 x 10(4), 3.1 x 10(6), and 4.1 x 10(6) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constants between the reduced enzyme and oxidized cytochrome b5 (k'3, M-1 min-1) were 4.3 x 10(8), 12 x 10(8), and 9.3 x 10(8) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constant between reduced hemoglobin and oxidized cytochrome b5 (k2) was 35 M-1 min-1 at pH 7.0. The theoretical Km for methemoglobin was 2.1 M at an infinite enzyme concentration at pH 7.0
一种可溶性红细胞细胞色素b5被纯化出来，作为高铁血红蛋白还原酶的底物和高铁血红蛋白的电子载体。该蛋白质的等电点为pH 4.3，在pH 7.0时E0'为-0.010。在pH 7.0以NADH作为电子供体时，该酶对这种蛋白质的Km值为1×10(-4) M，周转数（k5）为3.4×10(4) min-1。该酶对铁氰化物的最适pH为4.6，对细胞色素b5的最适pH为5.5，两种底物在pH 7至9时均有活性肩峰。通过考虑还原酶和细胞色素b5的E.S复合物，得到了表示高铁血红蛋白或细胞色素c还原的速率方程，该方程是高铁血红蛋白或细胞色素c、高铁血红蛋白还原酶和细胞色素b5的函数，并确定了相关的速率常数。在pH 7.0、pH 5.2和pH 5.0时，高铁血红蛋白与还原型细胞色素b5之间的速率常数（k1，M-1 min-1）分别为1.6×10(4)、3.1×10(6)和4.1×10(6)。在pH 7.0、pH 5.2和pH 5.0时，还原型酶与氧化型细胞色素b5之间的速率常数（k'3，M-1 min-1）分别为4.3×10(8)、12×10(8)和9.3×10(8)。在pH 7.0时，还原型血红蛋白与氧化型细胞色素b5之间的速率常数（k2）为35 M-1 min-1。在pH 7.0无限酶浓度下，高铁血红蛋白的理论Km值为2.1 M
