Aminotransferase activity and bioinformatic analysis of 1-aminocyclopropane-1-carboxylate synthase
Aminotransferase activity and bioinformatic analysis of 1-aminocyclopropane-1-carboxylate synthase
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DOI：
10.1021/bi002092a
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发表时间：
2000-12-12
期刊：
影响因子：
2.9
通讯作者：
Kirsch, JF
中科院分区：
文献类型：
--
作者：
Feng, L;Geck, MK;Kirsch, JF
磷酸吡哆醛（PLP）依赖性酶的机械命运在喹赛酮中间体之后发散。1-氨基环丙烷-1-羧酸（ACC）合酶是PLP依赖性酶的α家族的成员，其被优化以将电子从喹喔啉中间体引导至其底物S-腺苷-L-甲硫氨酸（SAM）的γ-碳，以产生ACC和5 '-甲硫基腺苷。所提供的数据表明，当其他氨基酸作为替代底物时，该醌也可以在辅因子的C-4'处接受质子以产生α-酮酸和磷酸吡哆胺（PMP）形式的酶。过量丙酮酸的加入将酶的PMP形式转化回PLP形式。通过NMR监测的溶剂交换显示从L-Ala的C-α-去质子化是可逆的，其速率比SAM的去质子化慢不到25倍。转氨作用的速率决定步骤是在奎宁中间体形成之后进行的。从酶结合的SAM中消除α，γ-的速率决定步骤同样显示在C-α-去质子化之后发生，并且喹喔啉中间体在该反应期间积累。BLAST检索、序列比对和结构比较表明ACC转肽酶与氨基转移酶在进化上相关。与先前发表的报告一致，发现PLP依赖性酶超家族的α和β家族之间不存在同源性。
The mechanistic fate of pyridoxal phosphate (PLP)-dependent enzymes diverges after the quinonoid intermediate. 1-Aminocyclopropane-1-carboxylate (ACC) synthase, a member of the alpha family of PLP-dependent enzymes, is optimized to direct electrons from the quinonoid intermediate to the gamma -carbon of its substrate, S-adenosyl-L-methionine (SAM), to yield ACC and 5'-methylthioadenosine. The data presented show that this quinonoid may also accept a proton at C-4' of the cofactor to yield alpha -keto acids and the pyridoxamine phosphate (PMP) form of the enzyme when other amino acids are presented as alternative substrates. Addition of excess pyruvate converts the PMP form of the enzyme back to the PLP form. C-alpha-deprotonation from L-Ala is shown by NMR-monitored solvent exchange to be reversible with a rate that is less than 25-fold slower than that of deprotonation of SAM. The rate-determining step for transamination follows the formation of the quinonoid intermediate. The rate-determining step for alpha,gamma -elimination from enzyme-bound SAM is likewise shown to occur after C-alpha-deprotonation, and the quinonoid intermediate accumulates during this reaction. BLAST searches, sequence alignments, and structural comparisons indicate that ACC synthases are evolutionarily related to the aminotransferases. In agreement with previously published reports, an absence of homology was found between the alpha and beta families of the PLP-dependent enzyme superfamily.