科研之友
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摘要
Urzymes short active core module derived from enzyme super families prepared from the two aminoacyltRNA synthetase (aaRS) clasSes contain only the modules shared by all related family members. They have been described as models :for ancestral forms. Understanding them,Currently Cleperids on inferences drawn from the crystal structures of the full-length enzymes. As aaRS Urzymes lack much of the mass of modern aaRS's,, retaining only a small portion of the hydrophobic cores of the full-length enzymes, it is desirable to characterize their structures. We report preliminary characterization of N-15 tryptophanyl-tRNA synthetase Urzyme by heteronuclear single - quantum-coherence '(HSQC) NMR spectroscopy supplemented by circular dichroism, thermal melting, and induced fluorescence of bound dye. The limited dispersion of 1H-chemical shifts (0.5 ppm.) is inconsistent A narrow ensemble offwell-packedstnictureS in either free or substrate -bound forms, although the number of resonances from the, bound state increases; indicating a modest, ligand-dependent gain in structure. Circular dichroism spectroscopy shows the presence of helices and evidence of cold denaturation, and all ligation states induce Sypro Orange fluorestence at ambient,temperatures. Although the term molten globure is difficult to define precisely, these characteristics are with most such definitions. Active-site titration shows that a majority of molecules retain. 60% of the transition state- stabilization free energy observed in modern synthetases. In, Contrast to the conventional view-that enzymes require stable tertiary structures, we conclude that a highly flexible,ground-state, ensemble can nevertheless bind tightly to the transition state for amino acid activation.
- 出版日期2016-6
