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

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.

Comparative Pharmacological Profiles of Various Bovine, Ovine, and Porcine Heparins

Unfractionated heparin is the first anticoagulant drug and has been successfully used clinically for over 80 years. Heparin and its analogues are used during surgery and dialysis and are often used to coat indwelling catheters and other devices where the vascular system is exposed. Most of the heparins used clinically are derived from porcine intestinal mucosa. However, heparins have also been manufactured from tissues of other mammalian species such as cows and sheep. Recently there have been attempts to generate bioengineered heparin in order to overcome contamination and antigenicity problems. Currently there are some concerns about the shortage of the porcine heparins as they are widely used in the manufacturing of the low-molecular-weight heparins. Moreover, due to cultural and religious reasons in some countries, alternative sources of heparins are needed. The Food and Drug Administration and other regulatory agencies have considered alternative sourcing of heparin for potential substitution of porcine heparin and are currently reviewing this matter. Numerous studies are ongoing to understand the structure-activity relationships of these various heparins. In this article, heparins from different animal sources were studied to determine the extent of biosimilarity between them. For these investigations, 10 batches each of bovine mucosal heparin (BMH), ovine mucosal heparin (OMH), and porcine mucosal heparin (PMH) were studied. These studies have demonstrated that OMH and PMH have comparable anticoagulant and antiproteases activities. However, BMH exhibited somewhat a lower potency compared to OMH and PMH in functional assays.