Malashkevich V N, Jäger J, Ziak M, Sauder U, Gehring H, Christen P, Jansonius J N
Abteilung Strukturbiologie, Universität Basel, Switzerland.
Biochemistry. 1995 Jan 17;34(2):405-14. doi: 10.1021/bi00002a004.
Chicken mitochondrial and Escherichia coli aspartate aminotransferases K258H, in which the active site lysine residue has been exchanged for a histidine residue, retain partial catalytic competence [Ziak et al. (1993) Eur. J. Biochem. 211, 475-484]. Mutant PLP and PMP holoenzymes and the complexes of the latter (E. coli enzyme) with sulfate and 2-oxoglutarate, as well as complexes of the mitochondrial apoenzyme with N-(5'-phosphopyridoxyl)-L-aspartate or N-(5'-phosphopyridoxyl)-L-glutamate, were crystallized and analyzed by means of X-ray crystallography in order to examine how the side chain of histidine 258 can substitute as a general acid/base catalyst of the aldimine-ketimine tautomerization in enzymic transamination. The structures have been solved and refined at resolutions between 2.1 and 2.8 A. Both the closed and the open conformations, identical to those of the wild-type enzyme, were observed, indicating that the mutant enzymes of both species exhibit the same conformational flexibility as the wild-type enzymes, although in AspAT K258H the equilibrium is somewhat shifted toward the open conformation. The replacement of the active site K258 by a histidine residue resulted only in local structural adaptations necessary to accommodate the imidazole ring. The catalytic competence of the mutant enzyme, which in the forward half-reaction is 0.1% of that of the wild-type enzyme, suggests that the imidazole group is involved in the aldimine-ketimine tautomerization. However, the imidazole ring of H258 is too far away from C alpha and C4' of the coenzyme-substrate adduct for direct proton transfer, suggesting that the 1,3-prototropic shift is mediated by a water molecule. Although there is enough space for a water molecule in this area, it has not been detected. Dynamic fluctuations of the protein matrix might transiently open a channel, giving a water molecule fleeting access to the active site.
鸡线粒体天冬氨酸氨基转移酶和大肠杆菌天冬氨酸氨基转移酶的K258H突变体（其中活性位点的赖氨酸残基已被组氨酸残基取代）仍保留部分催化活性[齐亚克等人（1993年），《欧洲生物化学杂志》211卷，475 - 484页]。为了研究组氨酸258的侧链如何替代作为酶促转氨作用中醛亚胺 - 酮亚胺互变异构的一般酸碱催化剂，对突变型磷酸吡哆醛（PLP）和磷酸吡哆胺（PMP）全酶以及后者（大肠杆菌酶）与硫酸盐和2 - 氧代戊二酸的复合物，以及线粒体脱辅基酶与N -（5'-磷酸吡哆醛基）-L - 天冬氨酸或N -（5'-磷酸吡哆醛基）-L - 谷氨酸的复合物进行了结晶，并通过X射线晶体学进行分析。这些结构已在2.1至2.8埃的分辨率下解析和精修。观察到了与野生型酶相同的封闭和开放构象，这表明两种物种的突变酶与野生型酶表现出相同的构象灵活性，尽管在天冬氨酸氨基转移酶K258H中，平衡略微向开放构象偏移。活性位点的K258被组氨酸残基取代仅导致为容纳咪唑环而必需的局部结构调整。突变酶在前向半反应中的催化活性是野生型酶的0.1%，这表明咪唑基团参与了醛亚胺 - 酮亚胺互变异构。然而，H258的咪唑环距离辅酶 - 底物加合物的Cα和C4'太远，无法直接进行质子转移，这表明1,3 - 质子转移是由水分子介导的。尽管该区域有足够的空间容纳一个水分子，但尚未检测到它。蛋白质基质的动态波动可能会暂时打开一个通道，使水分子短暂进入活性位点。
