Reactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase affinity alkylated by estrone 3-(bromoacetate): topographic studies with 16 alpha-(bromoacetoxy)estradiol 3-(methyl ether).
Reactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase affinity alkylated by estrone 3-(bromoacetate): topographic studies with 16 alpha-(bromoacetoxy)estradiol 3-(methyl ether).
复制标题
雌酮 3-（溴乙酸）烷基化的人胎盘 17 β,20 α-羟基类固醇脱氢酶亲和力的重新激活：用 16 α-（溴乙酰氧基）雌二醇 3-（甲醚）进行拓扑研究。
DOI：
10.1021/bi00341a014
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Strickler,RC
中科院分区：
文献类型：
--
作者：
Thomas,JL;LaRochelle,MC;Asibey-Berko,E;Strickler,RC
Estradiol 170-dehydrogenase and 20a-hydroxysteroid dehydrogenase, oxidoreductase activities copurified from the cytosol of human-term placenta as a homogeneous protein (native enzyme), were reactivated at equalrates to 100% activity following complete inactivation in the presence of cofactor (NADPH) with the affinity alkylator estrone 3-(bromoacetate). Reactivation was accomplished by base-catalyzed hydrolysis of steroidal ester-amino acid linkages in the enzyme active site. The rate of enzyme reactivation was pH dependent. In identical studies without NADPH, only 12% of the original enzyme activity was restored. Completely reactivated enzyme was repurified by dialysis. Enzyme in control mixtures (control enzyme) that contained estrone in place of alkylator was treated the same as the reactivated enzyme. Reactivated enzyme exhibited a 6.0-fold lower affinity for common substrates, a 1.8-fold lesser affinity for NAD+ and NADH, and the same affinity for NADP+ and NADPH compared to control enzyme. In incubations that included NADPH, the reactivated enzyme maintained full activity during a 20-h second exposure to estrone 3-(bromoacetate), but in identical incubations without NADPH, the reactivated enzyme was rapidly inactivated at the same rate as the control and native enzymes. The control and reactivated enzymes were inactivated at equalrates by 16a-(bromoacetoxy) estradiol 3-(methyl ether) in the presence or absence of cofactor (NADP+) and exhibited similar Kitz and Wilson inhibition constants for this affinity alkylator. Estrone 3-(bromo [2,-14C] acetate) incubated with native enzyme and NADPH produced ra-diolabeled 3-(carboxymethyl) histidine and S-(carboxymethyl) cysteine. 16a-(Bromo [2'-14C] acetoxy) estradiol 3-(methyl ether) produced twice the amount of radiolabeled 1, 3-bis (carboxymethyl) histidine and half the amounts of radiolabeled 3-(carboxymethyl) histidine and S-(carboxymethyl) cysteine in reactivated enzyme compared to control enzyme. The areas under the ninhydrin absorbancepeaks of 3-(carboxymethyl) histidine and S'-(carboxymethyl) cysteinefrom the radioalkylated reactivated, control, and native enzymes were compared. These results demonstrate that (1) the estradiol 170-dehydrogenase and 20a-hydroxysteroid dehydrogenase activities reside at a single site on one protein,(2) the active site histidine alkylated by estrone 3-(bromoacetate) in thepresence of NADPH, although not essential to catalytic activity, is involved in substrate binding, while cysteine alkylated in the absence of cofactor is essentialto catalysis and resides in the cofactor binding site,(3) diphosphopyridine and triphosphopyridine nucleotides occupy the enzyme active site in slightlydifferent orientations, and (4) 16a-(bromo [2'-14C] acetoxy) estradiol 3-(methyl ether) nonspecifically alkylates 3-(carboxymethyl) histidine in reactivated enzyme to produce radiolabeled 1, 3-bis (carboxymethyl) histidine and the two affinity alkylators modify common cysteinyl residues nonspecifically. e enzyme 170, 2Oa-hydroxysteroid dehydrogenase, an oxidoreductase purified from the cytosol of human-term pla-cental villous tissue, expresses both estradiol 170-de-hydrogenase and 20a-hydroxysteroid dehydrogenase activities (Purdy et al., 1964; Strickler & Tobias, 1980, 1982). Studies with affinity alkylators have suggested that the two activities reside at a single active site on one protein andhave modeled how progestin and estrogen substrates become oriented within the same enzyme active site (Strickler et al., 1981; Thomas et al., 1983). We now report reactivation of theenzyme activities following complete inactivation by the affinity al-kylator estrone 3 …
