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High‐Throughput Mass‐Spectrometry Monitoring for Multisubstrate Enzymes: Determining the Kinetic Parameters and Catalytic Activities of Glycosyltransferases

A novel high‐throughput screening (HTS) method with electrospray time‐of‐flight (ESI‐TOF) mass spectrometry allows i) rapid and broad screening of multisubstrate enzyme catalytic activity towards a range of donor and acceptor substrates; ii) determination of full multisubstrate kinetic parameters and the binding order of substrates. Two representative glycosyltransferases (GTs, one common, one recently isolated, one O‐glycosyltransferase (O‐GT), one N‐glycosyltransferase (N‐GT)) have been used to validate this system: the widely used bovine β‐1,4‐galactosyltransferase (EC 2.4.1.22), and the recently isolated Arabidopsis thaliana GT UGT72B1 (EC 2.4.1.‐). The GAR (green/amber/red) broad‐substrate‐specificity screen, which is based on the mass ion abundance of product, provides a fast, high‐throughput method for finding potential donors and acceptors from substrate libraries. This was evaluated by using six natural and non‐natural donors (α‐UDP‐D‐Glucose (UDPGlc), α‐UDP‐N‐Acetyl‐D‐glucosamine (UDPGlcNAc), α‐UDP‐D‐5‐thioglucose (UDP5SGlc), α‐GDP‐L‐fucose (GDPFuc), α‐GDP‐D‐mannose (GDPMan), α,β‐UDP‐D‐mannose (UDPMan)) and 32 broad‐ranging acceptors (sugars, plant hormones, antibiotics, flavonoids, coumarins, phenylpropanoids and benzoic acids). By using the fast‐equilibrium assumption, KM, kcat and KIA were determined for representative substrates, and these values were used to determine substrate binding orders. These screening methods applied to the two very different enzymes revealed some unusual substrate specificities, thus highlighting the utility of broad‐ranging substrate screening. For UGT72B1, it was shown that the donor specificity is determined largely by the nucleotide moiety. The method is therefore capable of identifying GT enzymes with usefully broad carbohydrate‐transfer ability.