Catalytic properties of enzymes used in biotechnology can be improved by eliminating those regulatory mechanisms that are not absolutely required for their functioning. We exploited mammalian glyceraldehyde-3-phosphate dehydrogenase as a model protein and examined the structural basis of the NAD(+) cooperative binding exhibited by its homologous isoenzymes: the somatic enzyme (GAPD) and the recombinant sperm-specific enzyme (dN-GAPDS). Moreover, we obtained a mutant dN-GAPDS, which misses the cooperativity, but exhibits a twofold increase in the specific activity instead (92 and 45 μmol NADH/min per mg protein for the mutant and the wild type proteins, respectively). Such an effect was caused by the disruption of the interdomain salt bridge D311-H124, which is located close to the active site of the enzyme. The thermal stability of the mutant protein also increased compared to the wild type form (heat absorption peak values were 70.4 and 68.6 °C, respectively). We expect our findings to be of importance for the purposes of biotechnological applications.

中文翻译：

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NAD结合合作性和精子特异的甘油醛-3-磷酸脱氢酶催化特性的结构基础。

通过消除那些功能并非绝对必需的调节机制，可以改善生物技术中所用酶的催化特性。我们利用哺乳动物甘油醛-3-磷酸脱氢酶作为模型蛋白，并研究了其同源同工酶：体细胞酶（GAPD）和重组精子特异性酶（dN-GAPDS）表现出的NAD（+）合作结合的结构基础。 。此外，我们获得了一个突变的dN-GAPDS，它缺少协同作用，但比活性却提高了两倍（突变体和野生型蛋白的每mg蛋白分别为92和45μmolNADH / min）。这种作用是由于域间盐桥D311-H124的破坏而引起的，该区域靠近酶的活性位点。与野生型相比，突变蛋白的热稳定性也有所提高（吸热峰值分别为70.4和68.6°C）。我们希望我们的发现对生物技术应用具有重要意义。