Kuravsky M L, Barinova K V, Asryants R A, Schmalhausen E V, Muronetz V I
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory 1/40, Moscow 119234, Russia.
Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Leninskie Gory 1/73, Moscow 119234, Russia.
Biochimie. 2015 Aug;115:28-34. doi: 10.1016/j.biochi.2015.04.016. Epub 2015 Apr 30.
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.
通过消除那些对其功能并非绝对必需的调节机制，可以提高生物技术中使用的酶的催化特性。我们利用哺乳动物甘油醛-3-磷酸脱氢酶作为模型蛋白，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））所表现出的NAD(+)协同结合的结构基础。此外，我们获得了一种突变型dN-GAPDS，它失去了协同性，但比活性却提高了两倍（突变型和野生型蛋白的比活性分别为每毫克蛋白92和45 μmol NADH/分钟）。这种效应是由位于酶活性位点附近的结构域间盐桥D311-H124的破坏引起的。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）。我们期望我们的发现对生物技术应用具有重要意义。
