REACTION-MECHANISM OF THIOREDOXIN - 3'-PHOSPHO-ADENYLYLSULFATE REDUCTASE INVESTIGATED BY SITE-DIRECTED MUTAGENESIS
REACTION-MECHANISM OF THIOREDOXIN - 3'-PHOSPHO-ADENYLYLSULFATE REDUCTASE INVESTIGATED BY SITE-DIRECTED MUTAGENESIS
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DOI：
10.1111/j.1432-1033.1995.347_1.x
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发表时间：
1995-10-01
期刊：
影响因子：
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通讯作者：
SCHWENN, JD
中科院分区：
文献类型：
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作者：
BERENDT, U;HAVERKAMP, T;SCHWENN, JD
研究了来自大肠杆菌的纯化重组腺苷3'-磷酸5'-磷酸硫酸盐（PADOPS）还原酶。减少硫氧还蛋白的迈克尔斯常数分别为23 mu m和10 mu m。该酶的V-MAX为94-99 Mu mol min（-1）mg（-1），分子活性/催化活性二聚体为95 s（-1）。腺苷3'，5'-双磷酸盐（PADOP）相对于Padops抑制竞争性（K-I 4 MU M）；腺苷2'，5'-双磷酸盐和亚硫酸盐不是抑制性的。 SH组抑制剂的烷基化不可逆地使酶失活。结构基因（CYSH）编码的小多肽，其单个Cys残基，位于保守的氨基酸簇（KXECGI/LH）中。通过位点特异性诱变研究了唯一的CYS和Tyr209参与将adpops还原为亚硫酸盐的参与：通过单链超过的延伸PCR突变Cysh；将突变的基因克隆在PBTAC1中，并在大肠杆菌RL 22（Delta cyshij）中表达。研究了同质Cys239ser和Tyr209phe突变垫还原酶的催化特性改变。单个CYS的突变使V-MAX的突变减少了4.5倍10（3）（V-Max，= 0.02-0.013 Mu mol min（-1）mg（-1）mg（-1）），对padops的K-M效应边缘效应（ 19m）和硫氧还蛋白（14 MU M）的硫氧还蛋白（14 mu M）突变，用硫氧还蛋白（K-M 1.5 mu M）急剧影响饱和并减少V-MAX（0.22-0.25 mu mol min（-1）mg（-1））除了adpops的K-M增加（31 mu M）外，还没有重组帕多普斯还原酶，作为假体基团。野生型和突变蛋白的还原形式与氧化形式之间的差异吸收光谱表明，除了Cys239和Tyr209外，未识别的TRP（Delta Lambda（Max）292 nm）似乎参与了还原。带有PADOPS的PING-PONG机制与理论型类型机理中酶异构体的减少酶异构体反应。
Properties of purified recombinant adenosine 3'-phosphate 5'-phosphosulfate (PAdoPS) reductase from Escherichia coli were investigated. The Michaelis constants for reduced thioredoxin and PAdoPS are 23 mu M and 10 mu M, respectively; the enzyme has a V-max of 94-99 mu mol min(-1) mg(-1) and a molecular activity/catalytically active dimer of 95 s(-1). Adenosine 3',5'-bisphosphate (PAdoP) inhibits competitively (K-i 4 mu M) with respect to PAdoPS; adenosine 2',5'-bisphosphate and sulfite are not inhibitory. Alkylation by SH-group inhibitors irreversibly inactivates the enzyme.The structural gene (cysH) encodes for a small polypeptide with a single Cys residue located in a conserved cluster (KXECGI/LH) of amino acids. Involvement of the only Cys and of Tyr209 in the reduction of PAdoPS to sulfite was investigated by site-specific mutagenesis: cysH was mutated by single-strand-overlay extension PCR; the mutated genes were cloned in pBTac1 and expressed in E. coli RL 22 (Delta cysHIJ). Homogenous Cys239Ser and Tyr209Phe mutant PAdoPS reductases were investigated for altered catalytic properties. Mutation of the single Cys reduced V-max by a factor of 4.5x 10(3) (V-max,, = 0.02-0.013 mu mol min(-1) mg(-1)) with marginal effects on K-m for PAdoPS (19 mu M) and reduced thioredoxin (14 mu M) Mutation of Tyr209 drastically affected saturation with thioredoxin (K-m 1.5 mu M) and decreased V-max (0.22-0.25 mu mol min(-1) mg(-1)) in addition to a small increase in K-m for PAdoPS (31 mu M).Chromophores as prosthetic groups were absent from recombinant PAdoPS reductase. Difference absorption spectra between reduced and oxidized forms of wild-type and mutated proteins indicated that, in addition to Cys239 and Tyr209, an unidentified Trp (Delta lambda(max) 292 nm) appears to be involved in the reduction.The data suggest a special ping-pong mechanism with PAdoPS reacting with the reduced enzyme isomer in a Theorell-Chance type mechanism.