Miyazaki K, Kadono S, Sakurai M, Moriyama H, Tanaka N, Oshima T
Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan.
Protein Eng. 1994 Jan;7(1):99-102. doi: 10.1093/protein/7.1.99.
3-Isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus HB8, was chemically modified with tetranitromethane which nitrated 1.5-2.0 Tyr residues per subunit. The nitration was biphasic and parallel to the loss of activity. The modified residue in the first phase was identified to be Tyr36, which is distantly located from the active site of the enzyme. The function of Tyr36 was investigated by site-specific replacement with Phe. The Michaelis constant for the substrate or co-enzyme was not altered by the replacement, whereas the catalytic constant decreased down to approximately 5%. X-ray analysis of the mutant enzyme revealed that Arg94 moved the largest distance among the active site residues, that is, the NH1 and NH2 of the guanidino group moved 1.11 and 1.32 A respectively. The results suggest that Arg94 is responsible for the enzyme catalysis.
来自嗜热栖热菌HB8（一种极端嗜热菌）的3-异丙基苹果酸脱氢酶，用四硝基甲烷进行化学修饰，每个亚基硝化1.5 - 2.0个酪氨酸残基。硝化反应呈双相，且与活性丧失平行。第一阶段被修饰的残基被鉴定为Tyr36，它距离酶的活性位点较远。通过用苯丙氨酸进行位点特异性置换来研究Tyr36的功能。底物或辅酶的米氏常数未因置换而改变，而催化常数降至约5%。突变酶的X射线分析表明，精氨酸94在活性位点残基中移动距离最大，即胍基的NH1和NH2分别移动了1.11 Å和1.32 Å。结果表明精氨酸94负责酶的催化作用。
