PLACENTAL 3B-HYDROXYSTEROID DEHYDROGENASE ISOMERASE
PLACENTAL 3B-HYDROXYSTEROID DEHYDROGENASE ISOMERASE
批准号:
2025112
负责人:
JAMES L THOMAS
金额:
$13.83万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
1985
资助国家:
美国
项目状态:
已结题
起止时间:
1985-08-01 至 1998-11-30
关键词:
Baculoviridae Insecta affinity labeling bacterial proteins chemical binding cofactor complementary DNA conformation enzyme activity enzyme mechanism enzyme substrate gene deletion mutation human tissue hydroxysteroid dehydrogenases molecular cloning nicotinamide adenine dinucleotide placenta point mutation pyridine nucleotide site directed mutagenesis steroid delta isomerase
中文摘要
本项目的目标是确定反应机理的
人胎盘3 β-羟基类固醇脱氢酶/类固醇5->4-烯-
异构酶(3 β-HSD/异构酶)。在
胎盘,3 β-HSD/异构酶催化母体转化
孕酮,一种促进子宫静止的激素
孕期酶竞争性地利用
脱氢表雄酮,胎儿肾上腺的主要类固醇产物
腺体近期,产生雄烯二酮,进一步代谢,
17 β-雌二醇。因此,胎盘3 β-HSD/异构酶桥接激素
母亲和胎儿之间的沟通,以调解当地
雌激素/孕激素平衡增加,
分娩的开始 3 β-HSD/异构酶的表征最终可能
允许胎盘酶的药理学控制,独立于
不同的性腺/肾上腺同工酶,以防止早产。
从人胎盘中纯化的均质酶已到手。野生型
这种酶在昆虫细胞中被杆状病毒过表达,
在动力学上与天然胎盘酶相同。基质的顺序
并研究了辅酶结合对3 β-HSD和异构酶活性的影响
使用经典的同位素配体交换和新的亲和性
标记/配体保护实验。胎盘异构酶反应
机制和激活的必要辅因子相比,已知的
细菌异构酶机制(无辅因子要求),通过测量
19-去甲睾酮和17 β-雌二醇结合后的光谱变化
在存在或不存在NADH的情况下与酶反应。停流
光谱实验解决了我们的假设,
构象变化介导NADH诱导的异构酶活化。
亲和放射性标记和配体保护实验绘制了结合
3 β-HSD底物、异构酶底物和辅因子的位点
这种单一多功能蛋白质的已知一级结构。 使用
我们的cDNA编码胎盘3 β-HSD/异构酶,可能催化
这些鉴定区域中的氨基酸使用修饰的
合成寡核苷酸。可能的辅因子和膜锚定
区域被删除。突变的和野生型cDNA通过
昆虫Sf-9细胞悬浮液中的杆状病毒。功能
每种表达的、纯化的突变体酶的显著性通过
先前应用于天然酶的严格动力学分析。这些
分析包括测量3 β的Michaelis-Menton常数,
HSD底物、异构酶底物和辅因子、抑制动力学
对于产物类固醇和NADH,以及使用
对修饰的结合位点具有特异性的亲和性烷化剂。这些
结构/功能研究定位了催化氨基酸,
一级结构中的酶活性,并验证我们独特的假设
连续的3 β-HSD和异构酶反应在
单个类固醇结合区中的连续位点。
英文摘要
The objective of this project is to define the reaction mechanisms of
human placental 3beta-hydroxysteroid dehydrogenase/steroid 5->4-ene-
isomerase (3beta-HSD/isomerase) by relating function to structure. In
placenta, 3beta-HSD/isomerase catalyzes the conversion of maternal
pregnenolone to progesterone, a hormone that promotes uterine quiescence
during pregnancy. The enzyme competitively utilizes
dehydroepiandrosterone, the primary steroid product of the fetal adrenal
gland near term, to produce androstenedione that is further metabolized to
17beta-estradiol. Thus, placental 3beta-HSD/isomerase bridges hormonal
communication between the mother and fetus to mediate the locally
increased estrogen/progesterone balance that has been associated with the
onset of labor. Characterization of 3beta-HSD/isomerase may ultimately
allow pharmacologic control of the placenta enzyme, independent of the
different gonadal/adrenal isoenzyme, to prevent premature births.
Homogeneous enzyme purified from human placenta is in-hand. Wild-type
enzyme as been overexpressed by baculovirus in insect cells and found to
be kinetically identical to native placenta enzyme. The order of substrate
and coenzyme binding for the 3beta-HSD and isomerase activities is studied
using both classic isotopic ligand exchange and novel affinity
labeling/ligand protection experiments. The placenta isomerase reaction
mechanism and activation by essential cofactor are compared to the known
bacterial isomerase mechanism (with no cofactor requirement) by measuring
spectral changes in 19-nortestosterone and 17beta-estradiol upon binding
to the enzyme in the presence or absence of NADH. Stopped-flow
spectroscopy experiments address our hypothesis that a time-dependent
conformational change mediates the NADH-induced activation of isomerase.
Affinity radiolabeling and ligand protection experiments map the binding
sites for 3beta-HSD substrate, isomerase substrate, and cofactor in the
known primary structure of this single, multifunctional protein. Using
our cDNA that encodes placental 3beta-HSD/isomerase, probable catalytic
amino acids in these identified regions are mutated using modified
synthetic oligonucleotides. Probable cofactor and membrane anchoring
regions are deleted. The mutated and wild-type cDNA are over-expressed by
baculovirus in suspensions of insect Sf-9 cells. The functional
significance of each expressed, purified mutant enzyme is determined by
rigorous kinetic analyses previously applied to the native enzyme. These
analyses include the measurement of Michaelis-Menton constants for 3beta-
HSD substrates, isomerase substrates, and cofactors, inhibition kinetics
for product steroids and NADH, and inactivation/protection profiles using
affinity alkylators that are specific for the modified binding site. These
structure/function studies localize the catalytic amino acids for the
enzyme activities in the primary structure and test our unique hypothesis
that the sequential 3beta-HSD and isomerase reactions are catalyzed at
contiguous sites in a single steroid binding region.
期刊论文(0)
专著(0)
科研奖励(0)
会议论文
Development and Application of In-Cell NMR Techniques
-
批准号:6505439
-
项目类别:
-
资助金额:$11.05万
-
财政年份:2002
-
负责人:JAMES L THOMAS
-
依托单位:
PLACENTAL 3 BETA-HYDROXYSTEROID DEHYDROGENASE/ISOMERASE
-
批准号:6636808
-
项目类别:
-
资助金额:$19.82万
-
财政年份:2000
-
负责人:JAMES L THOMAS
-
依托单位:
PLACENTAL 3 BETA-HYDROXYSTEROID DEHYDROGENASE/ISOMERASE
-
批准号:6520798
-
项目类别:
-
资助金额:$19.82万
-
财政年份:2000
-
负责人:JAMES L THOMAS
-
依托单位:
PLACENTAL 3 BETA-HYDROXYSTEROID DEHYDROGENASE/ISOMERASE
-
批准号:6130070
-
项目类别:
-
资助金额:$8.01万
-
财政年份:2000
-
负责人:JAMES L THOMAS
-
依托单位:
PLACENTAL 3 BETA-HYDROXYSTEROID DEHYDROGENASE/ISOMERASE
-
批准号:6363383
-
项目类别:
-
资助金额:$19.82万
-
财政年份:2000
-
负责人:JAMES L THOMAS
-
依托单位:
PLACENTAL 3 BETA-HYDROXYSTEROID DEHYDROGENASE/ISOMERASE
-
批准号:6348872
-
项目类别:
-
资助金额:$12.29万
-
财政年份:2000
-
负责人:JAMES L THOMAS
-
依托单位:
PLACENTAL 3B-HYDROXYSTEROID DEHYDROGENASE ISOMERASE
-
批准号:2197953
-
项目类别:
-
资助金额:$12.63万
-
财政年份:1985
-
负责人:JAMES L THOMAS
-
依托单位:
PLACENTAL 3 BETA-HYDROXYSTEROID DEHYDROGENASE/ISOMERASE
-
批准号:2403131
-
项目类别:
-
资助金额:$14.38万
-
财政年份:1985
-
负责人:JAMES L THOMAS
-
依托单位:
Placental 3B-Hydroxysteroid Dehydrogenase/Isomerase
-
批准号:6867021
-
项目类别:
-
资助金额:$23.2万
-
财政年份:1985
-
负责人:JAMES L THOMAS
-
依托单位:
Placental 3B-Hydroxysteroid Dehydrogenase/Isomerase
-
批准号:7012259
-
项目类别:
-
资助金额:$22.66万
-
财政年份:1985
-
负责人:JAMES L THOMAS
-
依托单位:
Placental 3B-Hydroxysteroid Dehydrogenase/Isomerase
-
批准号:7176164
-
项目类别:
-
资助金额:$22.0万
-
财政年份:1985
-
负责人:JAMES L THOMAS
-
依托单位:
Placental 3B-Hydroxysteroid Dehydrogenase/Isomerase
-
批准号:7348435
-
项目类别:
-
资助金额:$22.0万
-
财政年份:1985
-
负责人:JAMES L THOMAS
-
依托单位:
PLACENTAL 3B-HYDROXYSTEROID DEHYDROGENASE ISOMERASE
-
批准号:2197954
-
项目类别:
-
资助金额:$13.21万
-
财政年份:1985
-
负责人:JAMES L THOMAS
-
依托单位:
海外基金