STEROID BINDING SITES OF STEROID BINDING PROTEINS

本研究提案的长期目标是确定 催化剂所利用的催化机理的结构基础 来自两种细菌物种的δ-5-3-酮类固醇异构酶， 睾丸酮假单胞菌和恶臭假单胞菌。 使用 亲电和光化学亲和试剂， 溶液和作为与琼脂糖珠、残基 在结合的类固醇底物的A/B环的近端， 鉴定 将特别注意10 β试剂 被设计成与假定的质子反应的衍生物 被认为参与异构化反应的携带碱 由这些酶催化。 目前认为活性位点上的残留物以及 通过拟定亲和标记鉴定的其他残基 研究将使用寡核苷酸进行修改- 异构酶基因的定向诱变。 因此， 基因将被克隆，其核苷酸序列将由 将双脱氧方法应用于M13重组体。 突变基因会 在合适的宿主中表达并纯化突变蛋白 并测定其功能特性。 芳香族氨基酸残基的芳香族质子 受到类固醇结合的干扰，如通过H-NMR光谱所见， 将使用类似的诱变方法进行鉴定，其中 单个芳族残基被类似的脂族残基取代， 天然异构酶和突变异构酶NMR的比较 光谱制作

This research proposal's long-term objective is to determine the structural basis for the mechanism of catalysis utilized by the delta-5-3-ketosteroid isomerases from two bacterial species, Pseudomonas testosteroni and Pseudomonas putida. Using electrophilic and photochemical affinity reagents both in free solution and as solid phase conjugates with agarose beads, residues proximal to the A/B rings of bound steroid substrates will be identified. Particular attention will be paid to 10 beta reagent derivatives which are designed to react with the putative proton carrying base thought to be involved in the isomerization reaction catalyzed by these enzymes. Residues presently believed to be at the active site as well as additional residues identified by the proposed affinity labeling studies will be subjected to modification using oligonucleotide- directed mutagenesis of the isomerase gene. Thus, the isomerase gene will be cloned and its nucleotide sequence determined by the dideoxy method applied to M13 recombinants. The mutant genes will be expressed in a suitable host and the mutant proteins purified and their functional properties determined. The identity of aromatic amino acid residues whose aromatic protons are perturbed by steroid binding, as seen by H-NMR spectroscopy, will be identified using similar mutagenesis methodology in which individual aromatic residues are replaced by similar aliphatic residues and a comparison of native and mutant isomerase NMR spectra made.