STEROID BINDING SITES OF STEROID BINDING PROTEINS

这项研究提案的长期目标是确定 其催化作用机理的结构基础 来自两种细菌的Delta-5-3-酮类固醇异构酶， 假单胞菌和恶臭假单胞菌。vbl.使用 亲电和光化学亲和试剂均为游离态 溶液和AS与琼脂糖珠、残留物的固相结合 结合类固醇底物的A/B环的近端将是 已确认身份。将特别注意10种Beta试剂 旨在与假定的质子发生反应的衍生品 被认为参与异构化反应的载体碱 由这些酶催化。 目前认为在活性部位的残留物以及 通过拟议的亲和标记确定的其他残基 研究将使用寡核苷酸进行修改- 异构酶基因的定向突变。因此，异构酶 基因将被克隆，其核苷酸序列将由 对M13重组子采用双脱氧法。突变的基因将会 在合适的宿主中表达并纯化突变蛋白 并测定了它们的功能性质。 芳香质子所含芳香氨基酸残基的同一性 受到类固醇结合的干扰，如H-核磁共振波谱所示， 将使用类似的突变方法进行鉴定，其中 单个芳香族残基被相似的脂肪族取代 天然异构酶和突变异构酶的残基及其核磁共振比较 光谱制作完成。

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.