H-蛋白中Asp-68和Tyr-70定向突变对甘氨酸裂解酶系整体酶活的调控

还原甘氨酸途径被认为是最有前景的C1（one carbon）合成途径，其核心酶系是甘氨酸裂解酶系。在前期研究中，我们在甘氨酸裂解酶系H-蛋白“解开自保护”过程的研究中初步锁定了H-蛋白空腔内的潜在关键氨基酸残基为Ser-67、Asp-68和Tyr-70，并且证明Ser-67位点对甘氨酸酶系的整体酶活有重要影响。本文对H-蛋白的Asp-68和Tyr-70位点进行了侧链带正电突变（H-D68K、H-D68H、H-D68R和H-Y70K、H-Y70H、H-Y70R突变体），以及侧链非极性突变（H-D68G、H-D68V、H-D68M、H-D68L和H-Y70G、H-Y70V、H-Y70M、H-Y70L突变体），并测定了各突变体在甘氨酸裂解方向上的酶活。结果发现，Asp-68位带正电突变倾向降低甘氨酸酶系的整体酶活，Asp-68位非极性突变、Tyr-70位带正电突变及非极性突变在总体上倾向于维持或提升整体酶活。其中，相对野生型H-蛋白，H-D68R突变体的酶活下降了90.2%，H-Y70R、H-D68G和H-Y70L突变体的酶活分别提高了75.6%、53.6%和146%。硫辛酰胺与H-蛋白空腔内的氨基酸相互作用的分析结果表明，甘氨酸裂解酶系整体酶活的变化是由于H-蛋白的68和70位残基的突变阻碍或促进硫辛酰胺的释放。

Regulation of directed mutations of Asp-68 and Tyr-70 in H-protein on the overall enzyme activity of the glycine cleavage system

The reductive glycine pathway is considered to be the most promising one carbon (C1) synthesis pathway, and its core enzyme is the glycine cleavage system (GCS). In a previous study, we preliminarily identified the potential key amino acid residues in the H-protein cavity as Ser-67, Asp-68 and Tyr-70 in a study of the “unlocking self-protection” process of H-protein in the glycine cleavage system, and showed that the Ser-67 site had an important impact on the overall enzyme activity of the glycine cleavage system. In this paper, side-chain positively charged mutations (H-D68K, H-D68H, H-D68R and H-Y70K, H-Y70H, H-Y70R mutants) and side-chain nonpolar mutations (H-D68G, H-D68V, H-D68M, H-D68L and H-Y70G, H-Y70V, H-Y70M, H-Y70L mutants) were performed on the Asp-68 and Tyr-70 sites of H-protein, and the enzyme activities of each mutant in the glycine cleavage direction were determined. The results showed that positively charged mutations at Asp-68 tended to decrease the overall enzyme activity of the glycine cleavage system, while nonpolar mutations at Asp-68, positively charged mutations and nonpolar mutations at Tyr-70 tended to maintain or increase the overall enzyme activity. Compared with the wild-type H-protein, the enzyme activity of the H-D68R mutant decreased by 90.2%, and those of H-Y70R, H-D68G and H-Y70L mutant increased by 75.6%, 53.6% and 146%, respectively. An analysis of the interactions between lipoamide and residues in the cavity of H-protein showed that the change in the overall enzyme activity of the glycine cleavage system was due to the mutation at 68 and 70 residues of H-protein that hinders or promotes the release of lipoamide.