STRUCTURAL ANALYSIS OF SYNTHETICALLY ENGINEERED RNASES

X射线衍射分析将继续应用于单晶 一系列半合成牛胰腺核糖核酸酶， 改变催化效率或底物特异性， 进一步阐明天冬氨酸-121和 苯丙氨酸-120在建立催化能力和底物 这种酶的特异性。 母体完全活性的半合成酶 由残基1-118的非共价复合物组成，通过酶促获得 天然酶的消化，和含有残基的十四肽 111-124，通过化学合成获得。 在1.8A（R）处的精细结构 = 20.4）（Martin等人（1987）J. Biol. 262，15930-15938）。 如果asp-121被asn或ala取代，或 Phe-120被Leu取代后，催化效率降低了一个数量级 大小 这三种催化剂中的每一种的精制2.0-A结构 现在已经获得了有缺陷的类似物，但是对 酶活性丧失的结构基础被 在所有情况下发生的结构变化的多样性。 在 期望在未来的几年里， 活性位点配体的存在，我们计划进行结构分析 ASN-121和Leu-120类似物，特别是在 有效底物，2;-脱氧-2 '-氟尿苷基-3 '.5'-腺苷; 过渡态类似物，尿苷钒酸;和产物，3 '-胞苷酸。 一种半合成的类似物，其中his-119被非常接近的 电子等排的3-（3-吡唑基）-Ala部分没有活性。 扩散 因此，将真正的底物转化为这种类似物的晶体， 直接检测“酶-底物”复合物。 特别感兴趣的 是一系列核糖二核苷磷酸底物， 为了显示出27至3000秒-1的KCAT值， 酵素 如果用该酶获得的“酶-底物”复合物的结构 吡唑基类似物显示了用其他化合物未观察到的构象特征。 活性位点配体，含3-（3-吡唑基）-丙氨酸的双修饰类似物 在位置119和位置120的亮氨酸或位置121的天冬酰胺 如果可能的话，将进行制备、结晶，并在存在以下物质的情况下进行分析： 这些真实的基质。

X-ray diffraction analysis will continue to be applied to single crystals of a series of semisynthetic bovine pancreatic ribonucleases which exhibit altered catalytic efficiency or substrate specificity, with the intention of delineating further the roles played by aspartic acid-121 and phenylalanine-120 in establishing the catalytic power and substrate specificity of this enzyme. The parent, fully active semisynthetic enzyme consists of a non-covalent complex of residues 1-118, obtained by enzymatic digestion of the native enzyme, and a tetradecapeptide containing residues 111-124, obtained by chemical synthesis. A refined structure at 1.8 A (R = 20.4) of the parent complex has been obtained (Martin et al. (1987) J. Biol. Chem. 262, 15930-15938). If asp-121 is replaced by asn or ala, or phe-120 is replaced by leu, catalytic efficiency is reduced an order of magnitude. Refined 2.0-A structures of each of these three catalytically defective analogs have now been obtained, but interpretation of the structural basis for the loss of enzymatic activity is obscured by the multiplicity of structural changes that have occurred in all cases. In the expectation that more straightforward relationships will emerge in the presence of active site ligands, we plan to carry out structural analyses of the asn-121 and leu-120 analogs, in particular in the presence of the virtual substrate, 2;-deoxy-2'-fluorouridilyl-3'.5'-adenosine; the transition state analog, uridine vanadate; and the product, 3'-cytidylate. The semisynthetic analog in which his-119 is replaced by the very nearly isosteric 3-(3-pyrazolyl)-ala moiety is devoid of activity. Diffusion of true substrates into crystals of this analog should, therefore, permit direct examination of "enzyme-substrate" complexes. Of particular interest is a series of ribodinucleoside phosphate substrates that have been shown to exhibit kcat values ranging from 27 to 3000 sec-1 with the native enzyme. If the structures of the "enzyme-substrate" complexes obtained with the pyrazolyl analog reveal conformational features not observed with other active site ligands, double modified analogs containing 3-(3-pyrazolyl)-ala at position 119 and leucine at position 120 or asparagine at position 121 will be prepared, crystallized if possible, and analyzed in the presence of these true substrates.