大肠杆菌dethiobiotin合成酶的活性位点突变体:突变对酶催化的影响和结构属性。
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杨G, Sandalova T,洛曼K, Lindqvist Y, Rendina基于“增大化现实”技术
大肠杆菌dethiobiotin合成酶的活性位点突变体:突变对酶催化的影响和结构属性。
生物化学。1997年4月22日,36 (16):4751 - 60。
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9125495 (在PubMed]
- 文摘
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五个活性位点残基,Thr11、Glu12 Lys15, Lys37, Ser41,牵连的蛋白质晶体结构的研究大肠杆菌的防晒霜,在催化和衬底突变来确定其功能绑定。9个突变体酶、T11V E12A、E12D K15Q, K37L, K37Q, K37R, S41A,和S41C过度繁殖的大肠杆菌菌株缺乏同质性的功能内生dtb基因和净化。更换Thr11与缬氨酸增加了24000倍的公里(ATP)很少或根本没有改变Kd (ATP)公里(DAPA)和防晒霜k (cat),暗示这残留在稳态的重要作用为ATP亲和。两个Glu12突变体活动本质上是野生型的防晒霜(稍微升高k (cat))。与野生型的防晒霜,E12A有同样的明显公里(DAPA) subsaturating和饱和ATP浓度,表明可能的角色之间的绑定协同Glu12防卫事业厅和ATP。Lys15突变和Lys37导致失去催化活性(0.01% < 0.9%的野生型的防晒霜k (cat) K15Q Lys37突变体酶,分别)和提高两公里的DAPA(40倍和> 100倍高于野生型K15Q和Lys37突变体酶,分别)和ATP(1800倍和> 10倍高于野生型K15Q K37突变体酶,分别)。这些结果强烈表明Lys15和Lys37催化和底物结合是至关重要的。S41A和S41C本质上与野生型相同的k (cat),适度增加防卫事业厅和ATP公里和Kd (ATP)值。更换与半胱氨酸Ser41比与丙氨酸替代导致了更大的影响。这些数据表明之间的H-bond N7防卫事业厅和Ser41侧链对催化不是很重要。 The catalytic behavior of these mutant enzymes was also studied by pulse-chase experiments which produced results consistent with the steady-state kinetic analyses. X-ray crystallographic studies of four mutant enzymes, S41A, S41C, K37Q, and K37L, showed that the crystals were essentially isomorphous to that of the wild-type DTBS. The models of these mutant enzymes were well refined (1.9 -2.6 A) and showed good similarity to the wild-type enzyme (rmsd of C alpha atoms: 0.16-0.24 A). The crystal structure of S41C complexed with DAPA, Mn2+/Mg2+, and AMPPCP revealed a localized conformational change (rotations of side chains of Cys41 and Thr11) which can account for the changes in the kinetic parameters observed for S41C. The crystal structures of the Lys37 mutant enzymes showed that the positive charge of the side chain of Lys37 is indispensable. Mutations of Lys37 to either glutamine or leucine resulted in a shift of the metal ion (up to 0.5 A) together with side chains of other active site residues which could disrupt the subtle balance between the positive and negative charges in the active site. The conformational change of the phosphate binding loop (Gly8-X-X-X-X-X-Gly14-Lys15-Thr16) upon nucleotide binding observed previously [Huang, W., Jia, J., Gibson, K. J., Taylor, W. S., Rendina, A. R., Schneider, G., & Lindqvist, Y. (1995) Biochemistry 34, 10985] appears to be important to attain the proper active site scaffold.