CATALYSIS OF THE OXIDATIVE FOLDING OF RIBONUCLEASE-A BY PROTEIN DISULFIDE ISOMERASE - DEPENDENCE OF THE RATE ON THE COMPOSITION OF THE REDOX BUFFER
CATALYSIS OF THE OXIDATIVE FOLDING OF RIBONUCLEASE-A BY PROTEIN DISULFIDE ISOMERASE - DEPENDENCE OF THE RATE ON THE COMPOSITION OF THE REDOX BUFFER
复制标题
DOI：
10.1021/bi00217a004
复制
发表时间：
1991-01-22
期刊：
影响因子：
2.9
通讯作者：
GILBERT, HF
中科院分区：
文献类型：
--
作者：
LYLES, MM;GILBERT, HF
蛋白质二硫键异构酶(PDI)催化还原核糖核酸酶A氧化复性的速度强烈依赖于谷胱甘肽/谷胱甘肽二硫化物氧化还原缓冲液的组成。与非催化的谷胱甘肽介导的核糖核酸酶氧化折叠一样，PDI催化的反应的稳态速度相对于谷胱甘肽(GSH)和谷胱甘肽二硫键(GSSG)的浓度都表现出明显的最佳。当[GSH]=1.0 mM和[GSSG]=0.2 mM时，活性最好。在饱和核糖核酸酶浓度下的表观K(CAT)为0.46+/-0.05-mU-mo1(pDi的mU-mo1)-1，而非催化反应的表观一级速率常数为0.02+/-0.01min-1。GSH和GSSG浓度的变化对PDI催化和非催化反应的速度都有类似的影响，但在氧化程度较高的条件下，PDI的催化效率会降低。催化反应速度与非催化反应速度之比随着[GSH]2/[GSSG]量的增加而增加，并在[GSH]2/[GSSG]大于1 mM时接近一个恒定的极限值，这表明最佳活性需要还原的二硫醇形式的PDI。只要谷胱甘肽氧化还原缓冲液足够减少，以保持PDI的活性形式([GSH]2/[GSSG]&1 mm)，PDI提供的速率加速是合理的恒定的，尽管实际速率可能变化超过一个数量级。当[GSH]2/[GSSG]为0.06+/-0.01 mm时，PDI表现出最大速率加速度的一半。
The velocity of the oxidative renaturation of reduced ribonuclease A catalyzed by protein disulfide isomerase (PDI) is strongly dependent on the composition of a glutathione/glutathione disulfide redox buffer. As with the uncatalyzed, glutathione-mediated oxidative folding of ribonuclease, the steady-state velocity of the PDI-catalyzed reaction displays a distinct optimum with respect to both the glutathione (GSH) and glutathione disulfide (GSSG) concentrations. Optimum activity is observed at [GSH] = 1.0 mM and [GSSG] = 0.2 mM. The apparent K(cat) at saturating RNase concentration is 0.46 +/- 0.05-mu-mol of RNase renatured min-1 (mu-mol of PDI)-1 compared to the apparent first-order rate constant for the uncatalyzed reaction of 0.02 +/- 0.01 min-1. Changes in GSH and GSSG concentration have a similar effect on the rate of both the PDI-catalyzed and uncatalyzed reactions except under the more more oxidizing conditions employed, where the catalytic effectiveness of PDI is diminished. The ratio of the velocity of the catalyzed reaction to that of the uncatalyzed reaction increases as the quantity [GSH]2/[GSSG] increases and approaches a constant, limiting value at [GSH]2/[GSSG] greater than 1 mM, suggesting that a reduced, dithiol form of PDI is required for optimum activity. As long as the glutathione redox buffer is sufficiently reducing to maintain PDI in an active form ([GSH]2/[GSSG] > 1 mM), the rate acceleration provided by PDI is reasonably constant, although the actual rate may vary by more than an order of magnitude. PDI exhibits half of the maximum rate acceleration at a [GSH]2/[GSSG] of 0.06 +/- 0.01 mM.