Universal Glycosyltransferase Continuous Assay for Uniform Kinetics and Inhibition Database Development and Mechanistic Studies Illustrated on ST3GAL1, C1GALT1, and FUT1

Chemical systems glycobiology requires experimental and computational tools to make possible big data analytics benefiting genomics and proteomics. The impediment to tool development is that the nature of glycan construction and mutation is not template driven but rests on cooperative glycosyltransferase (GT) catalytic synthesis. What is needed is the collation of kinetics and inhibition data in a standardized form to make possible analytics of glycan and glycoconjugate synthesis, mechanism extraction, and pattern recognition. Currently, kinetics assays in use for GTs are not universal in processing nucleoside phosphate UDP, GDP, and CMP donor-based glycosylation reactions due to limitations in accuracy and large substrate volume requirements. Here we present a universal glycosyltransferase continuous (UGC) assay able to measure the declining concentration of the NADH reporter molecule through fluorescence spectrophotometry and, therefore, determine reaction rate parameters. The development and parametrization of the assay is based on coupling the nucleotide released from GT reactions with pyruvate kinase, via nucleoside diphosphate kinase (NDK) in the case of NDP-based donor reactions. In the case of CMP-based reactions, the coupling is carried out via another kinase, cytidylate kinase in combination with NDK, which phosphorylates CMP to CDP, then CDP to CTP. Following this, we conduct kinetics and inhibition assay studies on the UDP, GDP, and CMP-based glycosylation reactions, specifically C1GAlT1, FUT1, and ST3GAL1, to represent each class of donor, respectively. The accuracy of calculating initial rates using the continuous assay compared to end point (noncontinuous) assays is demonstrated for the three classes of GTs. The previously identified natural product soyasaponin1 inhibitor was used as a model to demonstrate the application of the UGC assay as a standardized inhibition assay for GTs. We show that the dose response of ST3GAL1 to a serial dilution of Soyasaponin1 has time-dependent inhibition. This brings into question previous inhibition findings, arrived at using an end point assay, that have selected a seemingly random time point to measure inhibition. Consequently, using standardized K_m values taken from the UGC assay study, ST3GAL1 was shown to be the most responsive enzyme to soyasaponin1 inhibition, followed by FUT1, then C1GALT1 with IC₅₀ values of 37, 52, and 886 μM respectively