The stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate is abolished in (Lys258----Ala) aspartate aminotransferase.
The stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate is abolished in (Lys258----Ala) aspartate aminotransferase.
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DOI：
10.1016/s0021-9258(18)60826-9
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发表时间：
1987-08
期刊：
影响因子：
--
通讯作者：
S. Kochhar;W. L. Finlayson;J. Kirsch;P. Christen
中科院分区：
文献类型：
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作者：
S. Kochhar;W. L. Finlayson;J. Kirsch;P. Christen
用[4 '-3H]吡哆胺5'-磷酸盐重构来自大肠杆菌的野生型脱天冬氨酸氨基转移酶导致pro-S C-4'-3H立体特异性释放到溶剂中。反应遵循一级动力学（t1/2 = 15分钟，pH 7.5和25 ℃），其速率常数与先前用来自鸡的线粒体天冬氨酸转氨酶发现的速率常数相似（Tobler，H. P.，克里斯滕，P.，和Gehring，H.（1986）J.Biol.Chem.261，7105-7108）。通过定点诱变用丙氨酸取代活性位点残基Lys 258产生无催化活性的酶（Malcolm，B.一、和Kirsch，J. F.（1985）Biochem. Biophys.通信资源132，915-921）。该突变酶不能从结合的[4 '-3H]吡哆胺5'-磷酸释放任何可测量的3 H。数据与早期的建议，赖氨酸258是必不可少的酮亚胺/醛亚胺互变异构，并证实了以前的结论，从酶结合的吡哆胺5 '-磷酸3 H交换机械对应于在转氨反应过程中的酮亚胺中间体的C-4'去质子化。
Reconstitution of wild-type apoaspartate aminotransferase from Escherichia coli with [4'-3H]pyridoxamine 5'-phosphate results in stereospecific release of the pro-S C-4' 3H to the solvent. The reaction follows first-order kinetics (t1/2 = 15 min at pH 7.5 and 25 degrees C), its rate constant being similar to that found previously with mitochondrial aspartate aminotransferase from chicken (Tobler, H.P., Christen, P., and Gehring, H. (1986) J. Biol. Chem. 261, 7105-7108). Substituting the active site residue Lys258 by alanine via site-directed mutagenesis yields a catalytically inactive enzyme (Malcolm, B. A., and Kirsch, J. F. (1985) Biochem. Biophys. Res. Commun. 132, 915-921). This mutant enzyme fails to release any measurable 3H from bound [4'-3H]pyridoxamine 5'-phosphate. The data are consistent with earlier proposals that Lys258 is indispensable for the ketimine/aldimine tautomerization, and corroborate the previous conclusion that 3H exchange from enzyme-bound pyridoxamine 5'-phosphate mechanistically corresponds to the deprotonation at C-4' of the ketimine intermediate during the transamination reaction.