Wikner C, Meshalkina L, Nilsson U, Nikkola M, Lindqvist Y, Sundström M, Schneider G
Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala.
J Biol Chem. 1994 Dec 23;269(51):32144-50.
A homologous expression system and a purification protocol for pure, highly active recombinant yeast transketolase have been developed. The invariant transketolase residue Glu418, which forms a hydrogen bond to the N-1' nitrogen atom of the pyrimidine ring of the cofactor thiamin diphosphate has been replaced by glutamine and alanine. Crystallographic analyses of the mutants show that these amino acid substitutions do not induce structural changes beyond the site of mutation. In both cases, the cofactor binds in a manner identical to the wild-type enzyme. Significant differences in the CD spectra of the mutant transketolases compared with the spectrum of wild-type enzyme indicate differences in the electron distribution of the aminopyrimidine ring of the cofactor. The E418Q mutant shows 2% and the E418A mutant shows about 0.1% of the catalytic activity of wild-type enzyme. The affinities of the mutant enzymes for thiamin diphosphate are comparable with wild-type transketolase. The hydrogen bond between the coenzyme and the side chain of Glu418 is thus not required for coenzyme binding but essential for catalytic activity. The results demonstrate the functional importance of this interaction and support the molecular model for cofactor deprotonation, the first step in enzymatic thiamin catalysis.
已开发出一种用于纯化、高活性重组酵母转酮醇酶的同源表达系统和纯化方案。转酮醇酶的不变残基Glu418与辅因子硫胺素二磷酸嘧啶环的N-1'氮原子形成氢键，已被谷氨酰胺和丙氨酸取代。突变体的晶体学分析表明，这些氨基酸取代不会在突变位点之外诱导结构变化。在这两种情况下，辅因子的结合方式与野生型酶相同。与野生型酶的光谱相比，突变型转酮醇酶的圆二色光谱存在显著差异，表明辅因子氨基嘧啶环的电子分布存在差异。E418Q突变体的催化活性为野生型酶的2%，E418A突变体的催化活性约为野生型酶的0.1%。突变酶对硫胺素二磷酸的亲和力与野生型转酮醇酶相当。因此，辅酶与Glu418侧链之间的氢键对于辅酶结合不是必需的，但对催化活性至关重要。结果证明了这种相互作用的功能重要性，并支持了辅因子去质子化的分子模型，这是酶促硫胺素催化的第一步。
