Lubini D G, Christen P
Proc Natl Acad Sci U S A. 1979 Jun;76(6):2527-31. doi: 10.1073/pnas.76.6.2527.
Paracatalytic enzyme modifications result from the oxidation of enzyme-substrate carbanions by extrinsic oxidants. During the oxidation of enzyme-activated substrates, transiently reactive intermediates are generated which, without being released from the enzyme, modify groups at the active site. For enzymes producing carbanion intermediates, the combination of the normal substrate with a suitable electron acceptor has thus been proposed as a highly specific binary system for their active site-directed modification. In this study, the structural features of paracatalytically modified fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from rabbit muscle have been elucidated. This enzyme is completely inactivated within 60 min in the presence of fructose 1,6-bisphosphate in saturating concentration and 0.5 mM hexacyanoferrate(III) (pH 7.6, 25 degrees C). The inactivation is caused by covalent incorporation of one triosephosphate derivative per subunit. Peptide analysis showed that the triosephosphate derivative forms an intrachain crosslink between lysine-146 and lysine-227. According to previous independent experimental evidence, both lysyl residues are located at the active site: the epsilon-amino group of lysine-227 forms a Schiff base intermediate with the carbonyl group of the substrate [Lai, C. Y., Nakai, N. & Chang, D. (1974) Science 183, 1204-1206] and alkylation of lysine-146 by the affinity labeling reagent N-bromoacetylethanolamine phosphate inactivates the enzyme [Hartman, F. C. & Brown, J. P. (1976) J. Biol. Chem. 251, 3057-3062]. The present data thus establish paracatalytic modification as a mode of active site-directed enzyme modification.
副催化酶修饰是由外在氧化剂氧化酶 - 底物碳负离子引起的。在酶激活的底物氧化过程中，会产生瞬态反应性中间体，这些中间体在不释放到酶外的情况下修饰活性位点的基团。因此，对于产生碳负离子中间体的酶，正常底物与合适电子受体的组合被提议作为一种用于其活性位点定向修饰的高度特异性二元体系。在本研究中，已阐明了来自兔肌肉的经副催化修饰的果糖 -1,6- 二磷酸醛缩酶（D- 果糖 -1,6- 二磷酸 D- 甘油醛 -3- 磷酸裂解酶，EC 4.1.2.13）的结构特征。在饱和浓度的果糖 1,6- 二磷酸和 0.5 mM 高铁氰化物（III）（pH 7.6，25℃）存在下，该酶在 60 分钟内完全失活。失活是由每个亚基共价掺入一个磷酸丙糖衍生物引起的。肽分析表明，磷酸丙糖衍生物在赖氨酸 -146 和赖氨酸 -227 之间形成链内交联。根据先前独立的实验证据，两个赖氨酰残基都位于活性位点：赖氨酸 -227 的 ε- 氨基与底物的羰基形成席夫碱中间体 [赖，C.Y.，中井，N. 和张，D.（1974 年）《科学》183，1204 - 1206]，并且亲和标记试剂 N- 溴乙酰乙醇胺磷酸对赖氨酸 -146 的烷基化使酶失活 [哈特曼，F.C. 和布朗，J.P.（1976 年）《生物化学杂志》251，3057 - 3062]。因此，目前的数据确立了副催化修饰作为一种活性位点定向酶修饰的模式。
