Human placental estradiol 17 beta-dehydrogenase: evidence for inverted substrate orientation ("wrong-way" binding) at the active site.
Human placental estradiol 17 beta-dehydrogenase: evidence for inverted substrate orientation ("wrong-way" binding) at the active site.
复制标题
人胎盘雌二醇 17 β-脱氢酶：活性位点底物方向反向（“错误方式”结合）的证据。
DOI：
10.1021/bi00412a036
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Sweet,F
中科院分区：
文献类型：
--
作者：
Murdock,GL;Warren,JC;Sweet,F
1988年2月18日收到修订稿件摘要：人胎盘雌二醇170-脱氢酶(EC1.1。1.62)与Ha-雌二醇17-(溴[2-14C]乙酸酯)(10微米)或170-雌二醇17-(溴[2-14C]乙酸酯)(微米)亲和标记。类固醇溴乙酸酯对该酶具有竞争性抑制作用，其K值分别为90微米(17溴乙酸酯)和134微米(170溴乙酸酯)。该酶的失活符合准一级动力学，t2=110min(17a溴乙酸酯)，tu2=220min(170溴乙酸酯)。两种溴乙酸酯的亲和放射性烷基化酶样品的氨基酸分析表明，N*-(羧基[14C]甲基)组氨酸是各自标记的修饰氨基酸。用胰酶消化产生的多肽通过反相高效液相色谱分离，发现含有TV*-(羧基[14C]甲基)组氨酸。17a溴乙酸酯和170溴乙酸酯都修饰了多肽Phe-Tyr-Gln-Tyr-Leu-Ala-His(ir-CM)-Ser-Lys中的相同组氨酸。以前，同样的组氨酸已经被雌酮3-(溴乙酸酯)独家标记，并且被证明不直接参与雌二醇D-环的催化氢转移。因此，推测该组氨酸位于结合的类固醇底物的A环附近。结果表明，雌二醇的17a溴乙酸酯和170溴乙酸酯D环类似物与雌酮A环类似物雌酮3(溴乙酸酯)与同一活性部位的组氨酸残基反应。此外，由于雌二醇17-(溴乙酸酯)都经历了相对于天然底物170-雌二醇活性部位的可逆结合步骤，雌二醇170-脱氢酶已在本实验室分离和结晶(Chin&Warren，1973；Chin等人，1976)。以前的研究旨在通过亲和标记技术确定酶活性部位的结构，使用了各种天然底物的溴乙酸酯类似物(Murdock&Warren，1982；Chin等，1982)。溴乙酸酯类似物是已证实的底物，因此必须在活性部位经历与天然底物类似的取向的可逆结合步骤。与的类似物
Revised Manuscript Received February 18, 1988 abstract: Human placental estradiol 170-dehydrogenase (EC 1.1. 1.62) was affinity labeled with Ha-estradiol 17-(bromo [2-14C] acetate)(10 µ) or 170-estradiol 17-(bromo [2-14C] acetate)(µ). The steroid bromoacetates competitively inhibit the enzyme (against 170-estradiol) with K¡ values of 90 µ (17 bromoacetate) and 134 µ (170 bromoacetate). Inactivation of the enzyme followed pseudo-first-order kinetics with a t\/2= 110 min (17a bromoacetate) and tu2= 220 min (170 bromoacetate). Amino acid analysis of the affinity radioalkylated enzyme samples from the two bromoacetates revealed that N*-(carboxy [14C] methyl) histidine was the modified amino acid labeled in each case. Digestion with trypsin produced peptides that were isolated by reverse-phase high-performanceliquid chromatography and found to contain TV*-(carboxy [14C] methyl) histidine. Both the17a bromoacetate and also the 170 bromoacetate modified the same histidine in the peptide Phe-Tyr-Gln-Tyr-Leu-Ala-His (ir-CM)-Ser-Lys. Previously, the same histidine had been exclusively labeledby estrone 3-(bromoacetate) and shown not to be directly involved in catalytic hydrogen transfer at the D-ring of estradiol. Therefore, this histidine was presumed to proximate the A-ring of the bound steroid substrate. The present results suggest that the 17a bromoacetate and 170 bromoacetate D-ring analogues of estradiol react with the same active site histidine residue as estrone 3-(bromoacetate), the A-ring analogue of estrone. Moreover, as each of the estradiol 17-(bromoacetates) undergoes the reversible binding step atthe enzyme relative to that of the natural substrate 170-estradiol active site.Estradiol 170-dehydrogenase has been isolated and crystallized in this laboratory (Chin & Warren, 1973; Chin et al., 1976). Previous studies aimed atdetermining the structure of the enzyme active site by affinity labeling techniques em-ployed various bromoacetate analogues of natural substrates (Murdock & Warren, 1982; Chin et al., 1982). The bromo-acetate analogues were proven substrates and, therefore, must undergo the reversible binding step at the active site in an orientation similar to the natural substrate. Analogues with