Chemoenzymatic synthesis of heparan sulfate (HS) and heparin represents an alternative to animal sourced material. In mammalian Golgi, d-glucuronyl C5-epimerase (Glce) is the first enzyme that acts on the heparin precursor N-sulfoheparosan (NSH), catalyzing the epimerization of d-glucuronic acid (GlcA) residues to l-iduronic acid (IdoA) residues, which are then sulfated by 2-O-sulfotransferase 1 (2OST-1) primarily on the IdoA residues. Despite the importance of Glce, the equilibrium and reaction kinetics of Glce have not been substantially investigated in isolation from 2OST-1. Glce catalyzes a reversible epimerization reaction with no change in molecular weight of NSH, thereby complicating rapid analysis by mass spectrometry. For this reason, we have developed a mechanistic assay to detect directly both the forward (IdoA from GlcA) and reverse (GlcA from IdoA) reactions by quantifying the chemical shift of the anomeric carbon on 2D 1H-13C HSQC and 1D 1H spectra. We have also used this assay in real time with pseudo 2D 1H –Time spectrum to measure the product formation over time. We have characterized steady-state kinetic parameters of Glce, including Vmax of 104 mmol/min and Km of 25.2 mM. We have also used this assay to pose more fundamental questions regarding the activity of the enzyme as a function of the NSH composition, specifically the NS content of the chain. We have observed increasing activity with increasing NS% on the polysaccharide that plateaus at approximately 35% conversion to IdoA. To elaborate further the processivity of Glce on the polysaccharide substrate, we have used epimerase mediated H-D exchange coupled with reactive oxygen species degradation of the polysaccharide to oligosaccharide components. This enabled a top-down analysis using LC-MS and determining the sequence specificity of Glce. This work is expected to result in a greater understanding of the various reaction parameters critical for large-scale chemoenzymatic synthesis of HS and heparin.

Characterization of the activity of glucuronyl C5-epimerase

2019 Spring National Meeting

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