来自巨型管虫（热液喷口管虫）营养体组织的ATP硫酸化酶。

ATP sulfurylase from trophosome tissue of Riftia pachyptila (hydrothermal vent tube worm).

作者信息

Renosto F, Martin R L, Borrell J L, Nelson D C, Segel I H

机构信息

Department of Biochemistry and Biophysics, University of California, Davis 95616.

出版信息

Arch Biochem Biophys. 1991 Oct;290(1):66-78. doi: 10.1016/0003-9861(91)90592-7.

DOI:10.1016/0003-9861(91)90592-7

PMID:1898101

Abstract

ATP sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) was extensively purified from trophosome tissue of Riftia pachyptila, a tube worm that thrives in deep ocean hydrothermal vent communities. The enzyme is probably derived from the sulfide-oxidizing bacteria that densely colonize the tissue. Glycerol (20% v/v) protected the enzyme against inactivation during purification and storage. The native enzyme appears to be a dimer (MW 90 kDa +/- 10%) composed of identical size subunits (MW 48 kDa +/- 5%). At pH 8.0, 30 degrees C, the specific activities (units x mg protein-1) of the most highly purified sample are as follows: ATP synthesis, 370; APS synthesis, 23; molybdolysis, 65; APSe synthesis or selenolysis, 1.9. The Km values for APS and PPi at 5 mM Mg2+ are 6.3 and 14 microM, respectively. In the APS synthesis direction, the Km values for MgATP and SO4(2-) are 1.7 and 27 mM, respectively. The Km values for MgATP and MoO4(2-) in the molybdolysis reaction are 80 and 150 microM, respectively. The Kia for MgATP is 0.65 mM. APS is a potent inhibitor of molybdolysis, competitive with both MgATP and MoO4(2-) (Kiq = 2.2 microM). However, PPi (+ Mg2+) is virtually inactive as a molybdolysis inhibitor. Oxyanion dead end inhibitors competitive with SO4(2-) include (in order of decreasing potency) ClO4- greater than FSO3- (Ki = 22 microM) greater than ClO3- greater than NO3- greater than S2O3(2-) (Ki's = 5 and 43 mM). FSO3- is uncompetitive with MgATP, but S2O3(2-) is noncompetitive. Each subunit contains two free SH groups, at least one of which is functionally essential. ATP, MgATP, SO4(2-), MoO4(2-), and APS each protect against inactivation by excess 5,5'-dithiobis-(2-nitrobenzoate). FSO3- is ineffective as a protector unless MgATP is present. PPi (+Mg2+) does not protect against inactivation. Riftia trophosome contains little or no "ADP sulfurylase." The high trophosome level of ATP sulfurylase (67-176 ATP synthesis units x g fresh wt tissue-1 from four different specimens, corresponding to 4-10 microM enzyme sites), the high kcat of the enzyme for ATP synthesis (296 s-1), and the high Km's for MgATP and SO4(2-) are consistent with a role in ATP formation during sulfide oxidation, i.e., the physiological reaction is APS + MgPPi in equilibrium SO4(2-) + MgATP.

摘要

ATP硫酸化酶（ATP：硫酸腺苷酰转移酶，EC 2.7.7.4）是从巨型管虫的营养体组织中大量纯化得到的，巨型管虫是一种生活在深海热液喷口群落中并茁壮成长的管虫。该酶可能来源于密集定殖在该组织中的硫化物氧化细菌。甘油（20% v/v）在纯化和储存过程中可保护该酶不被灭活。天然酶似乎是由相同大小的亚基（分子量48 kDa ± 5%）组成的二聚体（分子量90 kDa ± 10%）。在pH 8.0、30℃条件下，纯化程度最高的样品的比活性（单位×mg蛋白质⁻¹）如下：ATP合成，370；APS合成，23；钼酸分解，65；APSe合成或硒分解，1.9。在5 mM Mg²⁺存在下，APS和PPi的Km值分别为6.3和14 μM。在APS合成方向上，MgATP和SO₄²⁻的Km值分别为1.7和27 mM。钼酸分解反应中MgATP和MoO₄²⁻的Km值分别为80和150 μM。MgATP的Kia为0.65 mM。APS是钼酸分解的有效抑制剂，与MgATP和MoO₄²⁻均有竞争性（Kiq = 2.2 μM）。然而，PPi（+Mg²⁺）作为钼酸分解抑制剂实际上没有活性。与SO₄²⁻有竞争性的氧阴离子终产物抑制剂包括（按效力递减顺序）ClO₄⁻＞FSO₃⁻（Ki = 22 μM）＞ClO₃⁻＞NO₃⁻＞S₂O₃²⁻（Ki值分别为5和43 mM）。FSO₃⁻与MgATP无竞争性，但S₂O₃²⁻是非竞争性的。每个亚基含有两个游离的SH基团，其中至少一个在功能上是必需的。ATP、MgATP、SO₄²⁻、MoO₄²⁻和APS均可防止过量的5,5'-二硫代双（2-硝基苯甲酸）导致的失活。除非有MgATP存在，FSO₃⁻作为保护剂无效。PPi（+Mg²⁺）不能防止失活。巨型管虫营养体中几乎没有或没有“ADP硫酸化酶”。营养体中ATP硫酸化酶的高水平（来自四个不同标本的67 - 176个ATP合成单位×g新鲜湿重组织⁻¹，相当于4 - 10 μM酶位点）、该酶对ATP合成的高kcat（296 s⁻¹）以及对MgATP和SO₄²⁻的高Km值与硫化物氧化过程中ATP形成的作用一致，即生理反应是APS + MgPPi ⇌ SO₄²⁻ + MgATP处于平衡状态。