Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
Biochem Biophys Res Commun. 2010 Feb 5;392(2):212-6. doi: 10.1016/j.bbrc.2010.01.017. Epub 2010 Jan 12.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) catalyze similar enolase-type reactions. Both enzymes have a conserved non-catalytic Lys122 or Arg122 on the beta-strand E lying in the interface between the N- and C-terminal domains. We used site-directed mutagenesis to analyze the function of Lys122 in the form II Rhodospirillum rubrum RuBisCO (RrRuBisCO) and Bacillus subtilis RLP (BsRLP). The K122R mutant of RrRuBisCO had a 40% decrease in k(cat) for carboxylase activity, a 2-fold increase in K(m) for CO2, and a 1.9-fold increase in K(m) for ribulose-1,5-bisphosphate. K122M and K122E mutants of RrRuBisCO were almost inactive. None of the substitutions affected the thermal stability of RrRuBisCO. The K122R mutant of BsRLP had a 32% decrease in k(cat) and lower thermal stability than the wild-type enzyme. The K122M and K122E mutants of BsRLP failed to form a catalytic dimer. Our results suggest that the lysine residue is essential for function in both enzymes, although in each case, its role is likely distinct.
核酮糖-1,5-二磷酸羧化酶/加氧酶（RuBisCO）和 RuBisCO 样蛋白（RLP）催化类似的烯醇酶型反应。这两种酶在 N 端和 C 端结构域之间的界面上β-链 E 上都有保守的非催化性赖氨酸 122 或精氨酸 122。我们使用定点突变来分析 Rhodospirillum rubrum RuBisCO（RrRuBisCO）和枯草芽孢杆菌 RLP（BsRLP）中形式 II 的 Lys122 的功能。RrRuBisCO 的 K122R 突变体的羧化酶活性的 k(cat)降低了 40%，CO2 的 K(m)增加了 2 倍，核酮糖-1,5-二磷酸的 K(m)增加了 1.9 倍。RrRuBisCO 的 K122M 和 K122E 突变体几乎没有活性。这些取代均未影响 RrRuBisCO 的热稳定性。BsRLP 的 K122R 突变体的 k(cat)降低了 32%，热稳定性低于野生型酶。BsRLP 的 K122M 和 K122E 突变体未能形成催化二聚体。我们的结果表明，赖氨酸残基对于两种酶的功能都是必不可少的，尽管在每种情况下，其作用可能不同。
