Identification of residues involved in a conformational change accompanying substitutions for glutamate-43 in staphylococcal nuclease.
Identification of residues involved in a conformational change accompanying substitutions for glutamate-43 in staphylococcal nuclease.
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鉴定与葡萄球菌核酸酶中谷氨酸 43 取代相关的构象变化所涉及的残基。
DOI：
10.1021/bi00411a033
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Gerlt,JA
中科院分区：
文献类型：
--
作者：
Wilde,JA;Bolton,PH;Dell'Acqua,M;Hibler,DW;Pourmotabbed,T;Gerlt,JA
我们实验室最近的一篇论文[Hibler，D. W.，Stolowich，N. J.，雷诺兹，一、Gerlt，J. A.，Wilde，J. A.，&博尔顿，P. H.（1987）Biochemistry 26，6278]描述了在葡萄球菌核酸酶（SNase）的活性位点中产生推定的通用碱基Glu-43的定点取代，并使用1H NMR光谱来表征取代对突变蛋白质构象的影响。Glu-43（Asp，Gln，Asn，Ser和Ala）的替换都降低了催化效率，并改变了突变酶的一维和二维NMR光谱特性。我们已经准备和研究了几个样品的氘代野生型SNase，允许序列特异性共振分配几个芳香族和脂肪族氨基酸侧链的经验，在正常的一维光谱和二维NOESY光谱的变化的NMR光谱特性。由于质子化SNase的一维和二维光谱中共振的严重光谱拥挤，如果不是不可能的话，要在不对选定的氨基酸进行氘代的情况下获得分配是很困难的。我们已经获得的光谱表明，NOE强度的变化涉及空间上与两个苯丙氨酸残基相邻的缬氨酸残基;给定SNase的X射线结构[Cotton，F.一、哈岑，EE，& Legg，M. J.等人（1979）Proc. Acad. Sci. USA 76，2551]，这些残基必须是瓦尔-74、Phe-34和Phe-76。此外，在空间上与瓦尔-74和Phe-34相邻的经历NOE强度变化的亮氨酸残基可以被分配给Leu-25。我们还获得了光谱的突变酶与氨基酸取代这些和空间相邻的残基，揭示了显着的变化，在高场移动的甲基区域，从而支持我们的任务。这些分配表明，在活性位点中的残基43处的取代产生构象变化，该构象变化延伸到距离取代位置15（Leu-25）和30（Phe-76）之间的蛋白质分子区域中。现在关于SNase突变体中构象变化的程度和幅度的明确结论，其中活性位点残基已被定点诱变取代，强调了在定量解释SNase的作用时必须谨慎。（特异性或非特异性）氨基酸取代对突变酶的催化效率的影响。现在可以利用技术系统地改变一种酶的一级结构，因此，可以研究催化中的结构-功能关系（Ackers & Smith，1985; Leather巴罗& Fersht，1986; Gerlt，1987; Knowles，1987; Shaw，1987）。我们改变了葡萄球菌核酸酶（SNase）中43位的氨基酸，该氨基酸在野生型酶中是假定的谷氨酸
A recent paper from our laboratories [Hibler, D. W., Stolowich, N. J., Reynolds,. A., Gerlt, J. A., Wilde, J. A., & Bolton, P. H.(1987) Biochemistry 26, 6278] described the generation of site-directed substitutions for the putative general base Glu-43 in the active site of Staphylococcal nuclease (SNase) and the use of* H NMR spectroscopy to characterize the effect of the substitutions on the conformations of the mutant proteins. The replacements for Glu-43 (Asp, Gin, Asn, Ser, and Ala) both decreased the catalytic efficiency and changed the one-and two-dimensional NMR spectral properties of the mutant enzymes. We have prepared and studied the NMR spectral properties of several samples of deuteriated wild-type SNase that allowsequence-specific resonance assignments for several aromatic and aliphatic amino acid side chains that experience changes both in normal one-dimensional spectra and in two-dimensional NOESY spectra. Due to severe spectral congestion of resonances in the one-and two-dimensional spectra of protiated SNase, the assignments would havebeen difficult, if not impossible, to obtain without deuteriation of selected amino acids. The spectra we have obtained demonstrate that changes in NOE intensities involve a valine residue that is spatially adjacent to two phenylalanine residues; given the X-ray structure for SNase [Cotton, F. A., Hazen, EE, & Legg, M. J.(1979) Proc. Natl. Acad. Sci. USA 76, 2551], these residues must be Val-74, Phe-34, and Phe-76. In addition, a leucine residue experiencing changes in NOE intensities spatially adjacent to Val-74 and Phe-34 can be assigned to Leu-25. We have also obtained spectra of mutant enzymes with amino acid substitutions of these and spatially adjacent residues that reveal significant changes in the upfield-shifted methyl regions, thereby supporting our assignments. These assignments demonstrate that substitutions at residue 43 in the active site produce a conformational change that extends into a region of the protein molecule that is between 15 (Leu-25) and 30 Á (Phe-76) from the position of the substitution. This now unequivocal conclusion regarding the extent and magnitude of conformational changes in mutants of SNase in which active-site residues have been replaced by site-directed mutagenesis emphasizes the caution that must be exercised in quantitatively interpreting the effects of (specific or nonspecific) amino acid substitutions on the catalytic efficiency of mutated enzymes.Techniques are now available for the systematic alteration of the primary structure of an enzyme so that structure-function relationships in catalysis might be investigated (Ackers & Smith, 1985; Leather barrow & Fersht, 1986; Gerlt, 1987; Knowles, 1987; Shaw, 1987). We have altered the amino acid at position 43 in Staphylococcal nuclease (SNase), which in the wild-type enzyme is the putative glutamate
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DOI：
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发表时间：
1985
期刊：
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影响因子：
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