Smith H B, Larimer F W, Hartman F C
Protein Engineering and Molecular Mutagenesis Program, Oak Ridge National Laboratory, Tennessee.
Biochem Biophys Res Commun. 1988 Apr 29;152(2):579-84. doi: 10.1016/s0006-291x(88)80077-9.
Both activities of ribulose bisphosphate carboxylase/oxygenase are dependent on carbamylation by CO2 of a specific lysyl epsilon-amino group (Lys-191 of the enzyme from Rhodospirillum rubrum). To examine the stringency of the requirement for this lysyl side chain, Lys-191 was converted to an aminoethylcysteinyl residue (net replacement of a gamma-methylene group by a sulfur atom) by a combination of site-directed mutagenesis and subsequent chemical modification. The purified Cys-191 mutant was totally devoid of both carboxylase and oxygenase activities. However, this mutant protein exhibited tight-binding of the transition-state analogue, 2-carboxyarabinitol bisphosphate, a property heretofore ascribed solely to the carbamylated form of the carboxylase. Treatment of the mutant protein with ethylene imine restored catalytic activity to 4-7% of the wild-type level. The carboxylase:oxygenase activity ratio of the aminoethylated protein was unperturbed relative to that of wild-type enzyme.
1,5 - 二磷酸核酮糖羧化酶/加氧酶的两种活性均依赖于特定赖氨酸ε - 氨基（来自红螺菌的该酶的赖氨酸 - 191）被二氧化碳氨甲酰化。为了检验对该赖氨酸侧链需求的严格性，通过定点诱变和随后的化学修饰相结合的方法，将赖氨酸 - 191转变为氨乙基半胱氨酸残基（用一个硫原子净取代一个γ - 亚甲基）。纯化的半胱氨酸 - 191突变体完全没有羧化酶和加氧酶活性。然而，这种突变蛋白表现出对过渡态类似物2 - 羧基阿拉伯糖醇1,5 - 二磷酸的紧密结合，这一特性迄今为止仅归因于羧化酶的氨甲酰化形式。用乙撑亚胺处理突变蛋白可使催化活性恢复到野生型水平的4 - 7%。氨乙基化蛋白的羧化酶：加氧酶活性比相对于野生型酶未受影响。
