Aminolaevulinic acid synthase of Rhodobacter capsulatus: high-resolution kinetic investigation of the structural basis for substrate binding and catalysis.
Aminolaevulinic acid synthase of Rhodobacter capsulatus: high-resolution kinetic investigation of the structural basis for substrate binding and catalysis.
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荚膜红细菌的氨基乙酰丙酸合酶：底物结合和催化结构基础的高分辨率动力学研究
DOI：
10.1042/bj20121041
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Jahn D.
中科院分区：
文献类型：
--
作者：
Kaufholz;Hunter;Ferreira;Lendrihas;Hering;Jahn D.
血红素生物合成的第一个酶，ALAS（5-氨基乙酰丙酸合成酶），催化甘氨酸和琥珀酰CoA的吡哆醛5′-磷酸依赖性缩合为5-氨基乙酰丙酸、CO2和CoA。Rhodobacter capsulatusALAS的晶体结构提供了底物结合和催化的结构基础的第一个快照。为了阐明单个氨基酸残基在底物识别、底物定位、催化和结构蛋白重排的活性位点中的功能作用，产生了多个ALAS变体。在单次转换实验中观察到喹喔啉中间体I和II，表明存在α-氨基-β-氧代己二酸酯中间体。通过从产物5-氨基乙酰丙酸形成喹赛啉II的pH依赖性获得了进一步的证据。对Arg 21、Thr 83、Asn 85和Ile 86的功能进行了动力学分析。Arg 21、Thr 83和Ile 86都位于亚基间活性位点的第二个亚基上，是必需的。它们在第二亚基中的位置为两种底物的复杂缩合过程中所需的结构动力学提供了基础。ALAS变体T83 S对L-丙氨酸的利用表明，与相关氨基酸相比，该残基对于与甘氨酸底物结合的选择性的重要性。Asn 85被发现是唯一重要的琥珀酰辅酶A底物的识别和结合的选择性。本研究的结果提供了一个新的动态视图上的ALAS底物结合和催化的结构基础。
The first enzyme of haem biosynthesis, ALAS (5-aminolaevulinic acid synthase), catalyses the pyridoxal 5′-phosphate-dependent condensation of glycine and succinyl-CoA to 5-aminolaevulinic acid, CO2and CoA. The crystal structure ofRhodobacter capsulatusALAS provides the first snapshots of the structural basis for substrate binding and catalysis. To elucidate the functional role of single amino acid residues in the active site for substrate discrimination, substrate positioning, catalysis and structural protein rearrangements, multiple ALAS variants were generated. The quinonoid intermediates I and II were visualized in single turnover experiments, indicating the presence of an α-amino-β-oxoadipate intermediate. Further evidence was obtained by the pH-dependent formation of quinonoid II from the product 5-aminolaevulinic acid. The function of Arg21, Thr83, Asn85and Ile86, all involved in the co-ordination of the succinyl-CoA substrate carboxy group, were analysed kinetically. Arg21, Thr83and Ile86, all of which are located in the second subunit to the intersubunit active site, were found to be essential. Their location in the second subunit provides the basis for the required structural dynamics during the complex condensation of both substrates. Utilization ofL-alanine by the ALAS variant T83S indicated the importance of this residue for the selectiveness of binding with the glycine substrate compared with related amino acids. Asn85was found to be solely important for succinyl-CoA substrate recognition and selectiveness of binding. The results of the present study provide a novel dynamic view on the structural basis of ALAS substrate-binding and catalysis.
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影响因子：
11.4
作者：
Astner, I;Schulze, JO;Heinz, DW
通讯作者：
Heinz, DW
均质大肠杆菌 2-氨基-3-酮丁酸 CoA 连接酶（一种磷酸吡哆醛依赖性酶）的纯化、特性和 N 端氨基酸序列。
DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Mukherjee,JJ;Dekker,EE
通讯作者：
Dekker,EE
[52] 5-氨基乙酰丙酸合酶的纯化
DOI：
--
发表时间：
1986
期刊：
影响因子：
--
作者：
P. Jordan;A. Laghai
通讯作者：
A. Laghai
影响因子：
2.9
作者：
HUNTER, GA;FERREIRA, GC
通讯作者：
FERREIRA, GC
DOI：
10.1111/j.1432-1033.1973.tb03163.x
发表时间：
1973-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
FANICAGA.M;CLEMENTM.J
通讯作者：
CLEMENTM.J