Hamana H, Shinozawa T
Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Gunma, 376-8515, Japan.
J Biochem. 1999 Jan;125(1):109-14. doi: 10.1093/oxfordjournals.jbchem.a022246.
To investigate the role of the C-terminal region on the activity and thermostability of orotate phosphoribosyltransferase (OPRTase, EC 2. 4.2.10) from Thermus thermophilus, four C-terminal amino acid-deleted OPRTases (1, 2, 3, and 5 residues deleted) were constructed. The activities of all the mutant OPRTases were lower than that of wild-type OPRTase at all temperatures investigated (50-80 degreesC). V- and EV-OPRTase, mutants with Val and Glu-Val deletions, respectively, showed 63 to 75% of the activity of wild-type OPRTase at the temperatures investigated. EEV- and PLEEV-OPRTase, with Glu-Glu-Val and Pro-Leu-Glu-Glu-Val deletions, respectively, had activities of 22 to 35% of the wild-type. The Km values for orotate of all mutant OPRTases were more than 4-fold higher than that of the wild-type (25 microM). On the other hand, the Km for PRPP of the wild-type was 34 microM, and there were no significant differences between the wild-type and mutant OPRTases. The kcat values of the V- and EV-OPRTases were similar to that of the wild-type, but those of the EEV- and PLEEV-OPRTases were less than 50% that of the wild-type. The optimum temperature of all mutant OPRTases, 70 degreesC, was 10 degreesC lower than that of the wild-type. The remaining activities of wild-type and V-OPRTase after incubation at 90 degreesC for 20 min were 70 and 60% of the non-treated OPRTase activity, respectively. Although the remaining activity of EV-OPRTase was only 14% of the non-treated OPRTase activity, the addition of 200 mM KCl during heat treatment increased it to 70%. Circular dichroism spectroscopy revealed that V- and EV-OPRTase denature more easily than the wild-type OPRTase. The results suggest that the C-terminal valine and glutamic acid residues are important for the activity and thermostability of T. thermophilus OPRTase.
为了研究嗜热栖热菌乳清酸磷酸核糖转移酶（OPRTase，EC 2.4.2.10）C末端区域对其活性和热稳定性的作用，构建了4种C末端缺失氨基酸的OPRTase（分别缺失1、2、3和5个残基）。在所有研究温度（50 - 80℃）下，所有突变型OPRTase的活性均低于野生型OPRTase。V - OPRTase和EV - OPRTase分别为缺失Val和Glu - Val的突变体，在研究温度下其活性为野生型OPRTase的63%至75%。EEV - OPRTase和PLEEV - OPRTase分别缺失Glu - Glu - Val和Pro - Leu - Glu - Glu - Val，其活性为野生型的22%至35%。所有突变型OPRTase对乳清酸的Km值比野生型（25μM）高4倍以上。另一方面，野生型对PRPP的Km值为34μM，野生型和突变型OPRTase之间无显著差异。V - OPRTase和EV - OPRTase的kcat值与野生型相似，但EEV - OPRTase和PLEEV - OPRTase的kcat值不到野生型的50%。所有突变型OPRTase的最适温度为70℃，比野生型低10℃。野生型和V - OPRTase在90℃孵育20分钟后的剩余活性分别为未处理OPRTase活性的70%和60%。虽然EV - OPRTase的剩余活性仅为未处理OPRTase活性的14%，但热处理期间添加200 mM KCl可将其提高到70%。圆二色光谱显示，V - OPRTase和EV - OPRTase比野生型OPRTase更容易变性。结果表明，C末端的缬氨酸和谷氨酸残基对嗜热栖热菌OPRTase的活性和热稳定性很重要。
