Lyles M M, Gilbert H F
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Biochemistry. 1991 Jan 22;30(3):613-9. doi: 10.1021/bi00217a004.
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 kcat at saturating RNase concentration is 0.46 +/- 0.05 mumol of RNase renatured min-1 (mumol 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 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] greater than 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.
由蛋白质二硫键异构酶（PDI）催化的还原型核糖核酸酶A的氧化复性速度，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。与未催化的、由谷胱甘肽介导的核糖核酸酶氧化折叠一样，PDI催化反应的稳态速度在谷胱甘肽（GSH）和谷胱甘肽二硫化物（GSSG）浓度方面都呈现出明显的最佳值。在[GSH]=1.0 mM和[GSSG]=0.2 mM时观察到最佳活性。在核糖核酸酶浓度饱和时，表观催化常数kcat为0.46±0.05 μmol核糖核酸酶复性min-1（μmol PDI）-1，相比之下，未催化反应的表观一级速率常数为0.02±0.01 min-1。GSH和GSSG浓度的变化对PDI催化反应和未催化反应的速率有相似影响，但在采用的氧化性更强的条件下除外，此时PDI的催化效率会降低。催化反应速度与未催化反应速度的比值随着[GSH]2/[GSSG]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PDI的还原二硫醇形式是实现最佳活性所必需的。只要谷胱甘肽氧化还原缓冲液足够还原以将PDI维持在活性形式（[GSH]2/[GSSG]大于1 mM），PDI提供的速率加速就相当恒定，尽管实际速率可能相差一个数量级以上。在[GSH]2/[GSSG]为0.06±0.01 mM时，PDI表现出最大速率加速的一半。
