Trimboli A J, Quinn G B, Smith E T, Barber M J
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa 33612, USA.
Arch Biochem Biophys. 1996 Jul 1;331(1):117-26. doi: 10.1006/abbi.1996.0289.
Incubation of either Chlorella nitrate reductase or the recombinant flavin domain of spinach nitrate reductase with reagents specific for modification of cysteine residues, such as N-ethylmaleimide, resulted in a time-dependent inactivation of NADH:ferricyanide reductase activity which could be prevented by incubation in the presence of NADH. At 25 degrees C and employing a fixed enzyme:modifier ratio, the rate of inactivation for both the Chlorella and spinach enzymes followed the order p-chloromercuribenzoate > methyl methanethiosulfonate > 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid > N-ethylmaleimide. For the spinach flavin domain, inactivation by methyl methanethiosulfonate or p-chloromercuribenzoate was found to be concentration independent suggesting the absence of nonspecific modifications. Initial rate studies of the methyl methanethiosulfonate-modified flavin domain indicated a reduction in NADH:ferricyanide activity (Vmax) from 85 to 44 micromol NADH consumed/min/nmol FAD and an increase in the Km for NADH from 12 to 35 microM when compared to the native enzyme, confirming a role for cysteine residue(s) in maintaining diaphorase activity. Site-directed mutagenesis of the four individual cysteines (residues 17, 54, 62, and 240) in the recombinant spinach flavin domain resulted in mutant proteins with visible and CD spectra very similar to those of the wild-type domain. Initial rate studies indicated that only substitutions of serine for cysteine 240 decreased diaphorase activity with maximal NADH:ferricyanide activity for the C240S mutant corresponding to 51 micromol NADH consumed/min/nmol FAD with a Km for NADH of 14 microM. Mutation of C240 to Ala or Gly resulted in greater loss of activity. The thermal stability of the four serine mutants was slightly decreased compared to the wild-type domain with the C62S mutant exhibiting the greatest instability. In contrast to the effects on diaphorase activity, square wave voltammetric studies indicated changes in the oxidation-reduction midpoint potential for the FAD/FADH2 couple in the C54S (E0'= -197 mV), C62S (E0' = -226 mV), and C240S (E0' = -219 mV) mutants compared to the wild-type domain (E0' = -268 mV). These results indicate that of the four cysteine residues in the spinach nitrate reductase flavin domain, only C240 plays a role in maintaining diaphorase activity, while C54 has the greatest influence on flavin redox potential and that no correlation between changes in catalytic activity and flavin redox potential was observed.
将小球藻硝酸还原酶或菠菜硝酸还原酶的重组黄素结构域与对半胱氨酸残基具有修饰作用的试剂（如N - 乙基马来酰亚胺）一起温育，会导致NADH：铁氰化物还原酶活性随时间而失活，而在NADH存在下温育可防止这种失活。在25℃并采用固定的酶与修饰剂比例时，小球藻和菠菜酶的失活速率遵循以下顺序：对氯汞苯甲酸＞甲硫基甲烷磺酸盐＞2 -（4'-马来酰亚胺基苯胺基）萘 - 6 - 磺酸盐＞N - 乙基马来酰亚胺。对于菠菜黄素结构域，发现甲硫基甲烷磺酸盐或对氯汞苯甲酸引起的失活与浓度无关，这表明不存在非特异性修饰。对甲硫基甲烷磺酸盐修饰的黄素结构域的初始速率研究表明，与天然酶相比，NADH：铁氰化物活性（Vmax）从85微摩尔NADH消耗/分钟/纳摩尔FAD降低到44微摩尔，NADH的Km从12 microM增加到35 microM，这证实了半胱氨酸残基在维持递氢酶活性中的作用。对重组菠菜黄素结构域中四个单独的半胱氨酸（第17、54、62和240位残基）进行定点诱变，得到的突变蛋白的可见光谱和圆二色光谱与野生型结构域非常相似。初始速率研究表明，只有将第240位半胱氨酸替换为丝氨酸会降低递氢酶活性，C240S突变体的最大NADH：铁氰化物活性对应于51微摩尔NADH消耗/分钟/纳摩尔FAD，NADH的Km为14 microM。将C240突变为丙氨酸或甘氨酸会导致更大的活性丧失。与野生型结构域相比，四个丝氨酸突变体的热稳定性略有降低，其中C62S突变体表现出最大的不稳定性。与对递氢酶活性的影响相反，方波伏安法研究表明，与野生型结构域（E0' = -268 mV）相比，C54S（E0' = -197 mV）、C62S（E0' = -226 mV）和C240S（E0' = -219 mV）突变体中FAD/FADH2偶联的氧化还原中点电位发生了变化。这些结果表明，在菠菜硝酸还原酶黄素结构域的四个半胱氨酸残基中，只有C240在维持递氢酶活性中起作用，而C54对黄素氧化还原电位影响最大，并且未观察到催化活性变化与黄素氧化还原电位之间的相关性。
