Mutant aspartate aminotransferase (K258H) without pyridoxal-5'-phosphate-binding lysine residue. Structural and catalytic properties.
Mutant aspartate aminotransferase (K258H) without pyridoxal-5'-phosphate-binding lysine residue. Structural and catalytic properties.
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不含吡哆醛-5-磷酸结合赖氨酸残基的突变天冬氨酸转氨酶 (K258H)。
DOI：
10.1111/j.1432-1033.1993.tb17573.x
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
P. Christen
中科院分区：
文献类型：
--
作者：
M. Ziak;J. Jäger;V. Malashkevich;H. Gehring;R. Jaussi;J. Jansonius;P. Christen
如果天冬氨酸氨基转移酶的磷酸吡哆醛结合赖氨酸残基258被组氨酸残基交换，则酶保留部分催化能力[Ziak，M.，尧西河Gehring，H.和Christen，P.（1990）Eur. 187，329-333]。在高分辨率下测定了鸡线粒体和大肠杆菌的突变酶的三维结构。的多肽链的折叠模式被证明是相同的野生型酶，小的构象差异被限制在活性位点的部分。如果将天冬氨酸或谷氨酸加入到突变酶的吡哆醛形式中[λ max = 392 nm和330 nm（弱）;负CD在420 nm，正CD在370 nm和330 nm]，则外部醛亚胺（λ max = 430 nm;负CD在360 nm和430 nm）瞬时积累。在将2-酮戊二酸盐加入吡哆胺形式（λ max 330 nm，阳性CD）后，可以检测到推定的氯胺酮中间体;然而，使用乙酸异丙酯，在几秒钟内建立了外部醛亚胺和吡哆醛形式之间的平衡，这强烈有利于前者。与谷氨酸和谷氨酸的转氨循环进行只有三个数量级慢于野生型酶的整体反应。突变酶的比活性在25 ℃下为0.1 U/mg，在pH 6.0至8.5范围内保持恒定。用[4 '-3H]吡哆胺5'-磷酸盐重构突变脱辅基酶，导致3 H快速释放，一级速率常数kappa' = 5 × 10（-4）s-1，与野生型酶相似。显然，在天冬氨酸转氨酶中，组氨酸可以在一定程度上取代活性位点的赖氨酸残基。然而，H258的咪唑环似乎离C α和C4'太远而不能有效地在醛亚胺-氯胺酮互变异构化中充当质子供体/受体，这表明质子转移可能由居间的水分子介导。内部醛亚胺向外部醛亚胺的转移显然可以被后者的从头形成以及其在相反方向上的水解所取代。
If the pyridoxal-phosphate-binding lysine residue 258 of aspartate aminotransferase is exchanged for a histidine residue, the enzyme retains partial catalytic competence [Ziak, M., Jaussi, R., Gehring, H. and Christen, P. (1990) Eur. J. Biochem. 187, 329-333]. The three-dimensional structures of the mutant enzymes of both chicken mitochondria and Escherichia coli were determined at high resolution. The folding patterns of the polypeptide chains proved to be identical to those of the wild-type enzymes, small conformational differences being restricted to parts of the active site. If aspartate or glutamate was added to the pyridoxal form of the mutant enzyme [lambda max 392 nm and 330 nm (weak); negative CD at 420 nm, positive CD at 370 nm and 330 nm], the external aldimine (lambda max = 430 nm; negative CD at 360 nm and 430 nm) transiently accumulated. Upon addition of 2-oxoglutarate to the pyridoxamine form (lambda max 330 nm, positive CD), a putative ketamine intermediate could be detected; however, with oxalacetate, an equilibrium between external aldimine and the pyridoxal form, which was strongly in favour of the former, was established within seconds. The transamination cycle with glutamate and oxalacetate proceeds only three orders of magnitude more slowly than the overall reaction of the wild-type enzyme. The specific activity of the mutant enzyme is 0.1 U/mg at 25 degrees C and constant from pH 6.0 to 8.5. Reconstitution of the mutant apoenzyme with [4'-3H]pyridoxamine 5'-phosphate resulted in rapid release of 3H with a first-order rate constant kappa' = 5 x 10(-4) s-1 similar to that of the wild-type enzyme. Apparently, in aspartate aminotransferase, histidine can to some extent substitute for the active-site lysine residue. The imidazole ring of H258, however, seems too distant from C alpha and C4' to act efficiently as proton donor/acceptor in the aldimine-ketamine tautomerization, suggesting that the prototropic shift might be mediated by an intervening water molecule. Transmination of the internal to the external aldimine apparently can be replaced by de novo formation of the latter, and by its hydrolysis in the reverse direction.
天冬氨酸转氨酶的 K258R 突变体可稳定醌类中间体。
DOI：
--
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Toney,MD;Kirsch,JF
通讯作者：
Kirsch,JF
DOI：
10.1016/s0021-9258(18)60826-9
发表时间：
1987-08
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
S. Kochhar;W. L. Finlayson;J. Kirsch;P. Christen
通讯作者：
S. Kochhar;W. L. Finlayson;J. Kirsch;P. Christen
天冬氨酸转氨酶中战略赖氨酰残基的位点特异性甲基化。
DOI：
--
发表时间：
1988
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Roberts,WJ;Hubert,E;Iriarte,A;Martinez-Carrion,M
通讯作者：
Martinez-Carrion,M