A7699

To prepare thioester E2~Ub conjugates for assays, WT UbcH5b (100-200 μM) was incubated with E1 (1 μM) and ubiquitin (300-600 μM) at 37 °C for 40 minutes in 20 mM HEPES pH7.5, 50 mM NaCl, 2 mM MgCl₂, 2 mM TCEP (C4706, Sigma-Aldrich) and 10 mM ATP (A7699, Sigma-Aldrich). The reaction products were subsequently separated using a Superdex^TM 75 10/300 GL column equilibrated in 20 mM HEPES pH 7.5, 150 mM NaCl. The eluted conjugate was analysed by SDS-PAGE, and the fractions corresponding to the E2~Ub conjugate were pooled and concentrated to ~100 μM before snap-freezing.
Isopeptide linked UbcH5b~Ub conjugate for crystallography and crosslinking purposes was prepared using wild-type ubiquitin and UbcH5b, which included mutations to allow isopeptide conjugate formation (C85K), prevent backside binding (S22R) and increase E2 stability (C21S, C107S, C111S). The reaction contained E1 (1 μM), E2 (100–200 μM), ubiquitin (300–600 μM) and ATP (4 mM) (A7699, Sigma-Aldrich) in cycling buffer [25 mM Tris pH 9.5, 2.5 mM MgCl₂, 5 mM phosphocreatine (P7936, Sigma-Aldrich) and 0.6 U/mL creatine phosphokinase (C3755, Sigma-Aldrich)]. Typically a 5 mL reaction was incubated at 37 °C for 18-24 hours then separated on a HiLoad^TM 26/600 Superdex^TM 75 prep column, pre-equilibrated in 20 mM Tris-HCl pH 7.5, 200 mM NaCl^{47 (link)}.

For one
plate, ATP disodium salt (90 μL, 20 mM) (Sigma-Aldrich A7699)
and FAM-EGFR-derived peptide (15 μL, 100 μM) (LVEPLTPSGEAPNQ(K-5FAM)-COOH)
(Molecular Devices RP7129; reconstituted in Milli-Q H₂O
to a stock concentration of 100 μL; stored at −20°C)
were added to 2295 μL of a 1× assay buffer.

Enzymatic assays were performed using a modified protocol described previously^{37 (link),38 (link)}. Briefly, 2 µg of human recombinant GS obtained from Novus Biologicals (NBP2-52376) or purified GS obtained as described in the next section was added to the reaction mixture consisting of 100 mM imidazole/HCl (pH 7.2; I5513, Sigma-Aldrich), 50 mM glutamate (pH 7.2; G5889, Sigma-Aldrich), 20 mM ATP (pH 7.2; A7699, Sigma-Aldrich) and 20 mM MgCl₂ (M4880, Sigma-Aldrich). Unless otherwise indicated, 500 µl of reaction mixture was incubated for 2 min at 37 °C, and the reaction was initiated by adding NH₄Cl (A9434, Sigma-Aldrich), CH₃NH₂·HCl (M0505, Sigma-Aldrich), ¹⁵NH₄Cl (299251, Sigma-Aldrich) or ¹³C-methylamine (277630, Sigma-Aldrich) at the concentrations indicated in Fig. 4f,g or in the legends of Extended Data Fig. 3a,b. Aliquots (5 µl) of the reaction mixtures were sampled at the times specified in the figure legends, diluted 1:1,000 in LC–MS extraction solution and analyzed by LC–MS. K_m values were calculated in GraphPad 9.4 (Prism) by using a Michaelis–Menten equation. N²-Methyl-l-glutamine used in Fig. 2j was synthesized by replacing glutamate with 40 mM N-methyl-l-glutamate (ICN15555583, Fisher Scientific) in the reaction mixture described above. The reaction was incubated at 37 °C for 2 h, and an aliquot of the mixture was diluted 1:1,000 for LC–MS/MS analysis.