Hartman F C, Soper T S, Niyogi S K, Mural R J, Foote R S, Mitra S, Lee E H, Machanoff R, Larimer F W
J Biol Chem. 1987 Mar 15;262(8):3496-501.
Affinity labeling and comparative sequence analyses have placed Lys-166 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at the active site. The unusual nucleophilicity and acidity of the epsilon-amino group of Lys 166 (pKa = 7.9) suggest its involvement in catalysis, perhaps as the base that enolizes ribulosebisphosphate (Hartman, F.C., Milanez, S., and Lee, E.H. (1985) J. Biol. Chem. 260, 13968-13975). In attempts to clarify the role of Lys-166 of the carboxylase, we have used site-directed mutagenesis to replace this lysyl residue with glycine, alanine, serine, glutamine, arginine, cysteine, or histidine. All seven of these mutant proteins, purified by immunoaffinity chromatography, are severely deficient in carboxylase activity; the serine mutant, which is the most active, has a kcat only 0.2% that of the wild-type enzyme. Although low, the carboxylase activity displayed by some of the mutant proteins proves that Lys-166 is not required for substrate binding and argues that the detrimental effects brought about by amino acid substitutions at position 166 do not reflect gross conformational changes. As demonstrated by their ability to tightly bind a transition-state analogue (2-carboxyarabinitol 1,5-bisphosphate) in the presence of CO2 and Mg2+, some of the mutant proteins undergo the carbamylation reaction that is required for activation of the wild-type enzyme. Since Lys-166 is required neither for activation (i.e. carbamylation by CO2) nor for substrate binding, it must be essential to catalysis. When viewed within the context of previous related studies, the results of site-directed mutagenesis are entirely consistent with Lys-166 functioning as the base that initiates catalysis by abstracting the C-3 proton from ribulosebisphosphate. An alternative possibility that Lys-166 acts to stabilize a transition state in the reaction pathway cannot be rigorously excluded.
亲和标记和比较序列分析已将红螺菌中核酮糖二磷酸羧化酶/加氧酶的赖氨酸-166定位在活性位点。赖氨酸166的ε-氨基具有异常的亲核性和酸性（pKa = 7.9），这表明它参与催化作用，可能作为使核酮糖二磷酸烯醇化的碱（哈特曼，F.C.，米拉内兹，S.，和李，E.H.（1985年）《生物化学杂志》260，13968 - 13975）。为了阐明羧化酶中赖氨酸-166的作用，我们使用定点诱变将这个赖氨酰残基替换为甘氨酸、丙氨酸、丝氨酸、谷氨酰胺、精氨酸、半胱氨酸或组氨酸。通过免疫亲和层析纯化的所有这七种突变蛋白的羧化酶活性都严重不足；活性最高的丝氨酸突变体的kcat仅为野生型酶的0.2%。尽管一些突变蛋白显示出的羧化酶活性很低，但这证明赖氨酸-166不是底物结合所必需的，并且表明在位置166处的氨基酸取代所带来的有害影响并不反映总体构象变化。正如它们在二氧化碳和镁离子存在下紧密结合过渡态类似物（2 - 羧基阿拉伯糖醇1,5 - 二磷酸）的能力所证明的那样，一些突变蛋白经历了野生型酶激活所需的氨甲酰化反应。由于赖氨酸-166既不是激活（即由二氧化碳进行氨甲酰化）所必需的，也不是底物结合所必需的，它必定对催化作用至关重要。从先前相关研究的背景来看，定点诱变的结果完全符合赖氨酸-166作为通过从核酮糖二磷酸中提取C - 3质子来启动催化作用的碱的功能。赖氨酸-166在反应途径中起到稳定过渡态的另一种可能性不能被完全排除。
