Osumi-Davis P A, Sreerama N, Volkin D B, Middaugh C R, Woody R W, Woody A Y
Department of Biochemistry, Colorado State University, Fort Collins 80523.
J Mol Biol. 1994 Mar 18;237(1):5-19. doi: 10.1006/jmbi.1994.1205.
It has been demonstrated that the amino acids Asp537, Asp812, Lys631, His811 and Tyr639 are involved in bacteriophage T7 RNA polymerase catalysis. In the present paper, we report kinetic, spectroscopic and calorimetric characterization of the wild-type and mutant T7 RNA polymerases generated at these five loci (D537N, E; K631M, R; Y639F, S, A, W; H811Q, A; D812N, E). The wild-type enzyme has a substantial amount of secondary structure as determined by CD analysis (alpha-helix, 43%; beta-sheet, 14%; beta-turn, 25%; unordered, 18%). The CD spectra of 12 mutants at five loci are very similar to that of the wild-type, except for the mutant Y639W. Within experimental error, the thermal transition temperatures measured by CD and DSC as well as the lambda max values of the fluorescence spectra were the same for the wild-type and all of the mutants. Therefore, the overall folding and stability of the mutant enzymes are very similar to those of the wild-type enzyme, although small local conformational changes cannot be excluded. For the synthesis of the pentamer pppGGACU, the mutants D537E and D812E showed an approximately two- to threefold decrease in (kcat)app and an approximately two- to threefold increase in (Km)app, relative to the wild-type, in contrast to the mutants D537N and D812N which exhibited no detectable activity. The mutant K631R showed a sevenfold reduction in (kcat)app and a two- to threefold increase in (Km)app, supporting our earlier observation with the mutant K631M that Lys631 may be involved in phosphodiester bond formation. The mutant Y639S can synthesize the trimer GGA with an approximately 50-fold decrease in (kcat)app and a tenfold increase in (Km)app, relative to the wild-type, underlining the importance of the phenyl ring of Tyr639. The mutant H811A, in which the side-chain at position 811 is incapable of forming a hydrogen bond, can synthesize the trimer GGA with an approximately tenfold decrease in (kcat)app and an approximately 35-fold increase in (Km)app. Thus, either the hydrogen-bonding capacity of this residue is non-essential or some other group can functionally substitute for the His811 side-chain. The wild-type enzyme showed significant effects of the base position in the sequence on the apparent binding constants for the NTPs. The kinetics of GpG-primed trimer, tetramer and pentamer synthesis on three 22 bp templates were investigated for the wild-type and mutant enzymes with measurable activity.(ABSTRACT TRUNCATED AT 400 WORDS)
已证明氨基酸Asp537、Asp812、Lys631、His811和Tyr639参与噬菌体T7 RNA聚合酶的催化作用。在本文中，我们报告了在这五个位点（D537N、E；K631M、R；Y639F、S、A、W；H811Q、A；D812N、E）产生的野生型和突变型T7 RNA聚合酶的动力学、光谱学和量热学特征。通过圆二色性（CD）分析确定，野生型酶具有大量的二级结构（α-螺旋，43%；β-折叠，14%；β-转角，25%；无规卷曲，18%）。除了突变体Y639W外，五个位点的12个突变体的CD光谱与野生型非常相似。在实验误差范围内，通过CD和差示扫描量热法（DSC）测量的热转变温度以及荧光光谱的最大波长（λmax）值对于野生型和所有突变体都是相同的。因此，尽管不能排除小的局部构象变化，但突变酶的整体折叠和稳定性与野生型酶非常相似。对于五聚体pppGGACU的合成，相对于野生型，突变体D537E和D812E的表观催化常数（kcat）app降低了约两到三倍，表观米氏常数（Km）app增加了约两到三倍，这与未表现出可检测活性的突变体D537N和D812N形成对比。突变体K631R的表观催化常数（kcat）app降低了七倍，表观米氏常数（Km）app增加了两到三倍，这支持了我们早期对突变体K631M的观察结果，即Lys631可能参与磷酸二酯键的形成。相对于野生型，突变体Y639S可以合成三聚体GGA，其表观催化常数（kcat）app降低了约50倍，表观米氏常数（Km）app增加了十倍，这突出了Tyr639苯环的重要性。突变体H811A中811位的侧链不能形成氢键，它可以合成三聚体GGA，其表观催化常数（kcat）app降低了约十倍，表观米氏常数（Km）app增加了约35倍。因此，该残基的氢键结合能力要么是非必需的，要么是其他基团可以在功能上替代His811侧链。野生型酶显示序列中碱基位置对NTPs的表观结合常数有显著影响。研究了野生型和具有可测量活性的突变型酶在三个22 bp模板上进行GpG引发的三聚体、四聚体和五聚体合成的动力学。（摘要截断于400字）
