SUBSTRATE-SPECIFICITY AND VARIABLES AFFECTING EFFICIENCY OF MAMMALIAN FLAVIN ADENINE-DINUCLEOTIDE SYNTHETASE
SUBSTRATE-SPECIFICITY AND VARIABLES AFFECTING EFFICIENCY OF MAMMALIAN FLAVIN ADENINE-DINUCLEOTIDE SYNTHETASE
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DOI：
10.1021/bi00447a025
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发表时间：
1989-10-17
期刊：
影响因子：
2.9
通讯作者：
MCCORMICK, DB
中科院分区：
文献类型：
--
作者：
BOWERSKOMRO, DM;YAMADA, Y;MCCORMICK, DB
通过使用纯化的大鼠肝FAD合成酶（ATP：FMN腺苷酰转移酶，EC 2.7.7.2）评价底物特异性和产物抑制，所述合成酶通过使用优化的黄素亲和层析的改进的纯化方案获得。研究的FMN类似物分为三大类：在嘧啶环上取代和氮置换的FMN类似物、在苯环上取代的FMN类似物和具有N（10）侧链修饰的FMN类似物。嘧啶环上的取代和氮的置换对与酶的结合和FAD形成的影响最大。当3位的NH基团的氢键结合能力通过取代被阻断或去除时，这样的FMN类似物不充当酶的底物或抑制剂。苯环上被小基团取代似乎是可以容忍的，而较大的基团抑制结合。N（10）侧链的长度最佳为5个碳，并且对天然核糖基侧链具有最大的亲和力。亲和基质显示出相似的结合特性，即N（3）-（羧甲基）核黄素-琼脂糖不结合酶，而连接到黄素N（10）侧链的琼脂糖提供不同程度的纯化。C. dbd。2位的O基团、3位的NH基团和N（10）位的五碳侧链似乎对黄素底物与酶的结合最为关键。当存在足够的Mg 2+时，ATP以外的核苷三磷酸不作为底物或抑制剂。反应产物FAD和PPi作为ATP和FMN的抑制剂。发现FAD对ATP和FMN的Ki值分别为1.3和0.75 μ M。PPi对ATP和FMN的Ki值分别为200和66 μ M。
Substrate specificity and product inhibition have been evaluated by using purified rat liver FAD synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2), obtained by an improved purification protocol with optimized flavin affinity chromatography. FMN analogues studied fall into three general classifications: those with substitution on the pyrimidinoid ring and nitrogen replacement, those with substitution on the benzenoid ring, and those with N(10) side chain modifications. Substitutions on the pyrimidinoid ring and replacement of nitrogens have the greatest influence on binding to enzyme and FAD formation. When the hydrogen-bonding capacity of the NH group at position 3 is blocked or removed by substitution, such FMN analogues do not act as substrates or inhibitors of the enzyme. Substitutions on the benzenoid ring by small groups seem to be tolerated, while larger groups inhibit binding. Length of the N(10) side chain is optimal with five carbons and has greatest affinity for the natural ribityl side chain. Affinity matrices show similar binding characteristics in that the N(3)-(carboxymethyl)riboflavin-agarose does not bind enzyme, while agaroses linked to the flavin N(10) side chains provide varying degrees of purification. The C .dbd. O group at position 2, the NH group at position 3, and a five-carbon side chain at the N(10) position seem to be most crucial for flavin substrate binding to enzyme. Nucleoside triphosphates other than ATP do not act as substrates or inhibitors when sufficient Mg2+ is present. Products of the reaction, FAD and PPi, act as inhibitors against both ATP and FMN. Ki values for FAD against ATP and FMN were found to be 1.3 and 0.75 .mu.M, respectively. Ki values for PPi against ATP and FMN were 200 and 66 .mu.M, respectively.