Penning T M, Covey D F, Talalay P
Biochem J. 1981 Jan 1;193(1):217-27. doi: 10.1042/bj1930217.
Several steroid analogues containing conjugated acetylenic ketone groups as part of a seco-ring structure or as substituents on the intact steroid system are irreversible inhibitors of delta 5-3-oxo steroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Thus 10 beta-(1-oxoprop-2-ynyl)oestr-4-ene-3,17-dione (I), 5,10-seco-oestr-4-yne-3,10,17-trione (II), 17 beta-hydroxy-5,10-seco-oestr-4-yne-3,10-dione (III) and 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one (IV) irreversibly inactivate isomerase in a time-dependent manner. In all cases saturation kinetics are observed. Protection against inactivation is afforded by the powerful competitive inhibitor 19-nortestosterone. The inhibition constants (Ki) for 19-nortestosterone obtained from such experiments are in good agreement with those determined from conventional competitive-inhibition studies of enzyme activity. These compounds thus appear to be active-site directed. In every case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond probably had formed between the steroid and enzyme. Compound (I) is a very potent inhibitor of isomerase [Ki = 66.0 microM and k+2 = 12.5 x 10(-3) s-1 (where Ki is the dissociation constant of the reversible enzyme-inhibitor complex and k+2 is the rate constant for the inactivation reaction of the enzyme-inhibitor complex)] giving half-lives of inactivation of 30-45 s at saturation. It is argued that the basic-amino-acid residue that abstracts the intramolecularly transferred 4 beta-proton in the reaction mechanism could form a Michael-addition product with compound (I). In contrast, although compound (IV) has a lower inhibition constant (Ki = 14.5 microM), it is a relatively poor alkylating agent (k+2 = 0.13 x 10(-3) s-1). If the conjugated acetylenic ketone groups are replaced by alpha-hydroxyacetylene groups, the resultant analogues of steroids (I)-(IV) are reversible competitive inhibitors with Ki values in the range 27-350 microM. The enzyme binds steroids in the C19 series with functionalized acetylenic substituents at C-17 in preference to steroids in the C18 series bearing similar groups in the ring structure or as C-10 substituents. In the 5,10-seco-steroid series the presence of hydroxy groups at both C-3 and C-17 is deleterious to binding by the enzyme.
几种含有共轭炔酮基团作为开环结构一部分或完整甾体系统上取代基的甾体类似物，是睾丸酮假单胞菌中δ5 - 3 - 氧代甾体异构酶（EC 5.3.3.1）的不可逆抑制剂。因此，10β - (1 - 氧代丙 - 2 - 炔基)雌 - 4 - 烯 - 3,17 - 二酮（I）、5,10 - 开环雌 - 4 - 炔 - 3,10,17 - 三酮（II）、17β - 羟基 - 5,10 - 开环雌 - 4 - 炔 - 3,10 - 二酮（III）和17β - (1 - 氧代丙 - 2 - 炔基)雄 - 4 - 烯 - 3 - 酮（IV）以时间依赖性方式不可逆地使异构酶失活。在所有情况下均观察到饱和动力学。强效竞争性抑制剂19 - 去甲睾酮可提供对失活的保护作用。通过此类实验获得的19 - 去甲睾酮的抑制常数（Ki）与通过酶活性的传统竞争性抑制研究确定的常数高度一致。因此，这些化合物似乎是活性位点导向的。在每种情况下，失活的酶都可以进行透析而活性不会恢复，这表明甾体和酶之间可能形成了稳定的共价键。化合物（I）是异构酶的一种非常有效的抑制剂[Ki = 66.0 μM，k + 2 = 12.5×10⁻³ s⁻¹（其中Ki是可逆酶 - 抑制剂复合物的解离常数，k + 2是酶 - 抑制剂复合物失活反应的速率常数）]，在饱和时失活半衰期为30 - 45秒。有人认为，在反应机制中夺取分子内转移的4β - 质子的碱性氨基酸残基可能与化合物（I）形成迈克尔加成产物。相比之下，尽管化合物（IV）的抑制常数较低（Ki = 14.5 μM），但它是一种相对较差的烷基化剂（k + 2 = 0.13×10⁻³ s⁻¹）。如果共轭炔酮基团被α - 羟基乙炔基团取代，则所得的甾体（I） - （IV）类似物是可逆竞争性抑制剂，Ki值在27 - 350 μM范围内。该酶优先结合C19系列中在C - 17带有官能化炔取代基的甾体，而不是在环结构中或作为C - 10取代基带有类似基团的C18系列甾体。在5,10 - 开环甾体系列中，C - 3和C - 17处均存在羟基对酶的结合是有害的。
