Larson E M, Larimer F W, Hartman F C
Protein Engineering Program, Oak Ridge National Laboratory, Tennessee, USA.
Biochemistry. 1995 Apr 11;34(14):4531-7. doi: 10.1021/bi00014a005.
To evaluate the functions of flexible loop 6 at the active site of Rhodospirillum rubrum D-ribulose-1,5-bisphosphate carboxylase/oxygenase, the loop was truncated by cassette mutagenesis, whereby seven residues of the twelve-residue loop were excised and replaced by two glycyl residues. The purified loop-deletion mutant was totally devoid of carboxylase activity, but retained substantial catalytic competency in the enolization of ribulose bisphosphate (the initial step in the overall carboxylase pathway) and in normal processing of the six-carbon carboxylated intermediate (the terminal steps in the overall carboxylase pathway). Hence, catalytic impairment resides predominantly at the stage of carboxylation of the initial enediol(ate), a conclusion compatible with mechanistic deductions derived from crystallographic analyses. A critical role of loop 6 in the stabilization of the transition state for carboxylation is reinforced by the findings that the loop-deletion mutant displays preferentially compromised affinity for an analogue of the carboxylated intermediate relative to ribulose bisphosphate and that the mutant converts the substrate to a dicarbonyl compound as a consequence of beta-elimination of phosphate from the initial enediol(ate).
为了评估红螺菌1,5-二磷酸核酮糖羧化酶/加氧酶活性位点处柔性环6的功能，通过盒式诱变截短该环，切除了十二残基环中的七个残基，并用两个甘氨酰残基取代。纯化的环缺失突变体完全没有羧化酶活性，但在二磷酸核酮糖烯醇化反应（整个羧化酶途径的起始步骤）以及六碳羧化中间体的正常处理过程（整个羧化酶途径的终末步骤）中保留了相当的催化能力。因此，催化损伤主要存在于最初的烯二醇（盐）羧化阶段，这一结论与晶体学分析得出的机理推论相符。环6在羧化过渡态稳定中的关键作用因以下发现而得到加强：环缺失突变体相对于二磷酸核酮糖对羧化中间体类似物的亲和力优先受损，并且该突变体由于从最初的烯二醇（盐）中β-消除磷酸而将底物转化为二羰基化合物。
