Hartman F C, Lee E H
Biology Division, Oak Ridge National Laboratory, Tennessee 37831.
J Biol Chem. 1989 Jul 15;264(20):11784-9.
Diverse approaches that include site-directed mutagenesis have indicated a catalytic role of Lys-329 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. To determine whether Lys-329 is required for the initial enolization of ribulose bisphosphate or for some subsequent step in the overall reaction pathway, the competence of position 329 mutant proteins (devoid of carboxylase activity) in catalyzing exchange of solvent protons with the C-3 proton of substrate has now been examined. Irrespective of the amino acid substitution for Lys-329, the mutant protein retains 2-6% of the wild-type activity in the proton exchange reaction. The complete stability of ribulose bisphosphate during the enolization catalyzed by mutant protein suggests that the major effect of Lys-329 is to facilitate the addition of gaseous substrates (CO2 or O2) to the enediol intermediate. The exchange reaction requires Mg2+, is CO2-dependent, and is inhibited by the transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate. A mutant protein in which Lys-191, the site for carbamylation by CO2 in an obligatory activation step, is replaced by a cysteinyl residue totally lacks proton exchange activity. Barely detectable exchange activity (approximately 0.2% of wild-type) is displayed by the Lys-166----Cys mutant protein, consistent with the previously implicated role of Lys-166 in the deprotonation of ribulose bisphosphate. Retention of exchange activity by the Glu-48----Gln mutant protein, which is slightly active in overall carboxylation, demonstrates that active site Glu-48, like Lys-329, exerts its major effect at some step subsequent to the initial enolization.
包括定点诱变在内的多种方法表明，来自红螺菌的1,5 -二磷酸核酮糖羧化酶/加氧酶的赖氨酸-329具有催化作用。为了确定赖氨酸-329是1,5 -二磷酸核酮糖初始烯醇化反应所必需的，还是在整个反应途径中的某些后续步骤所必需的，现在已经研究了329位突变蛋白（缺乏羧化酶活性）催化溶剂质子与底物C-3质子交换的能力。无论赖氨酸-329被何种氨基酸取代，突变蛋白在质子交换反应中都保留了野生型活性的2% - 6%。在突变蛋白催化的烯醇化反应过程中，1,5 -二磷酸核酮糖完全稳定，这表明赖氨酸-329的主要作用是促进气态底物（CO₂或O₂）添加到烯二醇中间体上。交换反应需要Mg²⁺，依赖于CO₂，并被过渡态类似物2 -羧基阿拉伯糖醇1,5 -二磷酸抑制。在一个必需的激活步骤中，被CO₂氨甲酰化的位点赖氨酸-191被半胱氨酸残基取代的突变蛋白完全缺乏质子交换活性。赖氨酸-166→半胱氨酸突变蛋白显示出几乎检测不到的交换活性（约为野生型的0.2%），这与赖氨酸-166在1,5 -二磷酸核酮糖去质子化中先前涉及的作用一致。在整体羧化反应中略有活性的谷氨酸-48→谷氨酰胺突变蛋白保留了交换活性，这表明活性位点的谷氨酸-48与赖氨酸-329一样，在初始烯醇化反应后的某个步骤发挥其主要作用。
