产黄青霉ATP硫酸化酶的动力学和化学性质

Kinetic and chemical properties of ATP sulphurylase from Penicillin chrysogenum.

作者信息

Seubert P A, Grant P A, Christie E A, Farley J R, Segel I H

出版信息

Ciba Found Symp. 1979(72):19-47. doi: 10.1002/9780470720554.ch3.

DOI:10.1002/9780470720554.ch3

Abstract

Adenosine triphosphate sulphurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4.) has been purified from the filamentous fungus. Penicillium chrysogenum, and characterized physically, kinetically, and chemically. The P. Chrysogenum enzyme is an octomer (mol. wt. 440 000) composed of eight identical subunits (mol. wt. 55 000). Some physical constants are S20,w = 13.0 X 10(-13)s, D20,w = 2.94 X 10(-7) cm2 X s-1, v = 0.733 cm3 X g-1, A1%1cm = 8.71 at 278 nm. The enzyme catalyses (a) the synthesis of adenosine 5'-phosphosulphate (APS) and MgPPi from MgATP and SO2-4, (b) the hydrolysis of MgATP to AMP and MgPPi in the absence of SO2-4, (c) Mg32PPi-MgATP exchange in the absence of SO2-4, (d) molybdolysis of MgATP to AMP and MgPPi, (e) synthesis of MgATP and SO2-4 from APS and MgPPi, and (f) Mg32PPi-MgATP exchange in the presence of SO2-4. The Vmax values of reactions (a)-(c) are about 0.10-0.35 mumole X min-1 X mg enzyme-1. The Vmax values of reactions (d)-(f) are about 12-19 mumole X min-1 X mg enzyme-1. The catalytic activity of the enzyme in the direction of APS synthesis is rather low (0.13 unit X mg protein-1, corresponding to an active site turnover number of 7.15 min-1). However, the ATP sulphurylase content of mycelium growing on excess SO2-4 is 0.22 unit X g dry wt.-1, which is sufficient to account for the maximum in vivo rate of SO2-4 assimilation. The normal catalytic reaction is Ordered Bi Bi with A = MgATP, B = SO2-4, P = MgPPi, and Q = APS. Several lines of kinetic evidence suggest that the E.MgATP and E.APS complexes isomerize (to E approximately AMP.MgPPi and E approximately AMP.SO4, respectively) before the second substrate binds. Chemical modification studies have disclosed the presence of essential arginine, histidine, carboxyl, and tryosine residues. The latter is rather acidic (pKa = 7 or less). Nitration of the tyrosine increases the Km for MgATP without significantly affecting Kia for MgATP or Vmaxf. This result, and the fact that MgATP plus nitrate protects the enzyme against inactivation by tetranitromethane while MgATP alone does not, suggests that the essential tyrosine plays a role in nucleotide isomerization (perhaps as an adenylyl acceptor).

摘要

已从丝状真菌产黄青霉中纯化出三磷酸腺苷硫酸化酶（ATP：硫酸腺苷酰转移酶，EC 2.7.7.4.），并对其进行了物理、动力学和化学特性分析。产黄青霉的这种酶是一种由八个相同亚基（分子量55000）组成的八聚体（分子量440000）。一些物理常数为：沉降系数S20,w = 13.0×10⁻¹³ s，扩散系数D20,w = 2.94×10⁻⁷ cm²·s⁻¹，比容v = 0.733 cm³·g⁻¹，在278 nm处的吸光系数A1%1cm = 8.71。该酶催化：（a）由MgATP和SO₄²⁻合成腺苷5'-磷酸硫酸酯（APS）和MgPPi；（b）在无SO₄²⁻时MgATP水解为AMP和MgPPi；（c）在无SO₄²⁻时Mg³²PPi - MgATP交换；（d）MgATP的钼解作用生成AMP和MgPPi；（e）由APS和MgPPi合成MgATP和SO₄²⁻；（f）在有SO₄²⁻时Mg³²PPi - MgATP交换。反应（a） - （c）的Vmax值约为0.10 - 0.35微摩尔·分钟⁻¹·毫克酶⁻¹。反应（d） - （f）的Vmax值约为12 - 19微摩尔·分钟⁻¹·毫克酶⁻¹。该酶在APS合成方向上的催化活性相当低（0.13单位·毫克蛋白⁻¹，对应活性位点转换数为7.15分钟⁻¹）。然而，在过量SO₄²⁻上生长的菌丝体中ATP硫酸化酶含量为0.22单位·克干重⁻¹，这足以解释SO₄²⁻同化的最大体内速率。正常的催化反应是有序的双双反应，其中A = MgATP，B = SO₄²⁻，P = MgPPi，Q = APS。几条动力学证据表明，在第二个底物结合之前，E·MgATP和E·APS复合物会异构化（分别异构化为E≈AMP·MgPPi和E≈AMP·SO₄）。化学修饰研究揭示了存在必需的精氨酸、组氨酸、羧基和酪氨酸残基。后者相当酸性（pKa = 7或更低）。酪氨酸的硝化增加了对MgATP的Km值，而对MgATP的Kia或Vmaxf没有显著影响。这一结果，以及MgATP加硝酸盐可保护该酶免受四硝基甲烷失活而单独的MgATP则不能这一事实，表明必需的酪氨酸在核苷酸异构化中起作用（可能作为腺苷酰受体）。