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.
