Elucidating the unusual reaction kinetics of D-glucuronyl C5-epimerase

Abstract

The chemoenzymatic synthesis of heparin, through a multienzyme process, represents a critical challenge in providing a safe and effective substitute for this animal-sourced anticoagulant drug. D-glucuronyl C5-epimerase (C5-epi) is an enzyme acting on a heparin precursor, N-sulfoheparosan, catalyzing the reversible epimerization of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). The absence of reliable assays for C5-epi has limited elucidation of the enzymatic reaction and kinetic mechanisms. Real time and offline assays are described that rely on 1D 1H NMR to study the activity of C5-epi. Apparent steady-state kinetic parameters for both the forward and the pseudo-reverse reactions of C5-epi are determined for the first time using polysaccharide substrates directly relevant to the chemoenzymatic synthesis and biosynthesis of heparin. The forward reaction shows unusual sigmoidal kinetic behavior, and the pseudo-reverse reaction displays nonsaturating kinetic behavior. The atypical sigmoidal behavior of the forward reaction was probed using a range of buffer additives. Surprisingly, the addition of 25 mM each of CaCl2 and MgCl2 resulted in a forward reaction exhibiting more conventional Michaelis–Menten kinetics. The addition of 2-O-sulfotransferase, the next enzyme involved in heparin synthesis, in the absence of 3′-phosphoadenosine 5′-phosphosulfate, also resulted in C5-epi exhibiting a more conventional Michaelis–Menten kinetic behavior in the forward reaction accompanied by a significant increase in apparent Vmax. This study provides critical information for understanding the reaction kinetics of C5-epi, which may result in improved methods for the chemoenzymatic synthesis of bioengineered heparin.

Polysaccharide Sequence Influences the Specificity and Catalytic Activity of Glucuronyl C5-Epimerase

“…NSH with 1 and 9% GlcNAc residues resulted in ∼32% IdoA residues, consistent with the "true" thermodynamic equilibrium between GlcA and IdoA. 9,10,13 When the GlcNAc residue content was increased to 52%, the product of C5-epi catalysis contained only 8% IdoA residues. Moreover, C5-epi was inactive on the fully Nacetylated heparosan, consistent with the literature.…”

“…The C5-epi shown in Figure 5 is based on a homology model of the engineered enzyme used in this study against the 3D structure of C5-epi in zebra fish. 9 In the case of NSH without GlcNAc residues, C5-epi acts on one GlcA residue at a time converting it to IdoA but infrequently acts on two adjacent GlcA residues. This information obtained using gel permeation chromatography after complete heparanase BP digestion results in hexasaccharides, represented by the dashed arrows present in Figure 5.…”

“…4,5 C5-epi catalyzes a reversible reaction converting glucuronic acid (GlcA) to iduronic acid (IdoA), resulting in a polysaccharide with an equilibrium mixture of ∼70% GlcA and 30% IdoA. 9 Thus, NSH chemically produced from heparosan consists of three different monosaccharide units, GlcA, N-sulfoglucosamine (GlcNS), and GlcNAc. 4−6 The polydispersity and structural complexity of NSH offers a significant challenge in understanding the mechanism of action of C5-epi on this substrate, particularly the impact (i.e., the percentage and distribution of GlcNAc residues) on carbohydrate sequence on the epimerization reaction as it proceeds.…”

“…The mixture was incubated for 17 h to allow for the epimerization reaction to attain apparent equilibrium. 9 The reaction was then terminated by heat shocking the enzyme at 95 °C for 10 min and centrifuged at 12 000 rcf for 10 min to separate the aggregated enzyme, and the product was lyophilized for further analysis. The assay was run in biological quintuplicates.…”

Physiology and Pathophysiology of Heparan Sulfate in Animal Models: Its Biosynthesis and Degradation

“…Glucuronic acid C5-epimerase (GLCE) is a unique enzyme that catalyzes the epimerization of glucuronic acid to iduronic acid [138]. This conversion occurs by catalyzing the isomerization of an equatorial COOH in glucuronic acid at C5 to an axial COOH in iduronic acid.…”