Fig. 3: Role of the second metal binding site (Mn²⁺_N) in catalysis.

a Electron density for Mn²⁺_N is absent in crystals grown in the absence of Mn²⁺. b Soaking apo-TxgGalNAc-T3 crystals in Mn²⁺ results in the appearance of Mn²⁺_N, where the Fo-Fc omit map is contoured at 6σ (orange) and the anomalous signal (data collection λ =1 Å) is contoured at 3σ (pink). c There are no differences in the sidechain conformations of the metal binding residues in the apo structure (no Mn²⁺) and the co-crystal complex containing di-glycopeptide substrate. d–f The second metal influences catalysis by regulating Michaelis complex formation (E:S) in a pH sensitive manner. d Conformation of the site in the crystal structure at pH 9.5. Deprotonated sidechains bind tightly to Mn²⁺ and GalNAc, forming a tight Michaelis complex and inhibiting product release. e Quantum chemistry calculations at pH 7.0, where His deprotonation promotes ES complex formation, and His protonation promotes product release. f Quantum chemistry calculations where the His333 sidechain is fully protonated. Here, His333 is no longer binding Mn²⁺, and Glu554 interactions with GalNAc are reduced, disrupting ES complex formation and catalysis. Quantum chemistry generated coordinate files are included as Supplementary Dataset 1.