. Author manuscript; available in PMC: 2020 Aug 28.

Published in final edited form as: J Am Chem Soc. 2019 Aug 16;141(34):13442–13453. doi: 10.1021/jacs.9b04695

Table 1.

Kinetic Parameters of Wild-Type and Engineered GalNAc-T2 and UDP-Sugar Pairs^a

T2/UDP-Sugar	k_cat (s⁻¹)	K_m (μM)	k_cat/K_m (mM⁻¹S⁻¹)
WT-T2/1	0.813 ± 0.017	30 ± 2	28
T2(I253A/L310A)/(S)-3	0.566 ± 0.014	43 ± 4	13
T2(I253A/L310A)/7	0.61 ± 0.03	160 ± 20	3.8
T2(I253A/L310A)/11	0.68 ± 0.02	56 ± 6	12
T2(I253A/L310A)/(S)-12	0.84 ± 0.05	430 ± 50	2.0
T2(I253A/L310A)/13	0.158 ± 0.003	2.6 ± 0.8	61

To determine K_m and k_cat values for UDP-GalNAc and UDP-GalNAc analogs, initial rates were measured by incubating wild-type or double mutant GalNAc-T2 with concentrations of UDP-sugars varying from 15.6 to 500 μM and with a constant concentration of acceptor peptide (Peptide-1 = 267 μM for 1, (S)-3, 11; Peptide-1 = 250 μM for 7, (S)-12, 13). The glycosylation was conducted at 37 °C, and three aliquots were taken within 15 min and quenched by addition of aqueous EDTA (150 mM, pH = 8.0). Products were quantified by HPLC separation and peak integration. Enzymatic kinetic parameters were obtained by nonlinear regression fitting using GraphPad Prism. All data represent the mean of technical triplicates, and error depicts the standard deviation.