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[Preprint]. 2023 Oct 19:2023.02.01.526536. Originally published 2023 Feb 1. [Version 3] doi: 10.1101/2023.02.01.526536

Fig. 4. S. cerevisiae Trm10-KRR has similar substrate and cosubstrate binding affinities as the wild-type enzyme but lacks catalytic activity.

Fig. 4.

Trm10 C-terminal domain structure (PDB: 4JWJ) shown as A, cartoon and B, electrostatic surface potential highlighting the lysine (K) and arginine (R) residues substituted with glutamic acid in the Trm10-KRR variant. C, Fluorescence anisotropy determination of substrate tRNAGly-GCC binding affinities (KD) for wild-type Trm10 (black circles; KD = 20 ± 4 nM) and Trm10-KRR (blue squares; KD = 34 ± 5 nM). Results shown are for three independent assays plotted together and fit to equation 1, as described in the Experimental Procedures. D, Thin-layer chromatography methylation assay to determine methylation efficiency of wild-type Trm10 and Trm10-KRR with substrate tRNAGly-GCC. Reactions contained 10-fold serial dilutions of purified enzyme, as indicated by triangles, or no enzyme (−). Representative isothermal titration calorimetry (ITC) analysis of wild-type Trm10 and Trm10-KRR binding to E, SAM cosubstrate and F, methylation reaction by-product SAH. Binding affinities derived from fits to both replicates are given in Supplemental Table S1.