Mechanistic studies of glycoside hydrolase substrates and inhibitors

Glycoside hydrolases (GHs) are the enzymes that catalyze the cleavage of glycosidic bonds, which link carbohydrate units to other biomolecules. The work in this thesis comprises mechanistic studies of various GHs with experimental and computational methods. In Chapter 2, the transition state (TS) for the hydroxide-catalyzed hydrolysis of 4-nitrophenyl α-d-mannopyranoside in aqueous media was studied via kinetic isotope effect (KIE) measurements and computational methods. The findings were consistent with a mechanism involving formation of a transient oxirane intermediate. This mechanism supports the pre- viously proposed neigboring group participation mechanism for a GH99 mannosidase. Chapter 3 and 4 provides a mechanistic analysis of a GH36 α-galactosidase mechanism-based covalent inhibitor. Crystal structures of the enzyme-bound species demonstrate that the Michaelis complexes for intact inhibitor and product have half-chair conformations, while the covalent intermediate adopts a flattened half-chair conformation. QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into active site interactions. TSs for covalent intermediate formation and hydrolysis were assessed using experimental KIEs and QM/MM calculations. The enzyme was found to stabilize TS charge development on a remote C5-allylic center of the reacting carbasugar, and catalysis proceeds via a loose SN 2 TS with no discrete cationic intermediate. In chapter 5 KIEs were measured for the hydrolysis of α-d-glucopyranosyl fluoride by two inverting GHs and compared to values computed with multiscale QM/MM methods for the hydrolysis of α-d-glucopyranosyl fluoride promoted by an inverting Aspergillus niger GH15 α-glucosidase to give β-d-glucopyranose. KIEs were also measured for catalysis of β-d-glucopyranosyl fluoride by the Trichoderma virens GH55 inverting β-glucosidase; this reaction occurs via the "Hehre resynthesis–hydrolysis mechanism" to give the hydrolysis product α-d-glucopyranose. The TSs for both reactions are essentially identical with fluoride ion departure occurring with active site stabilization of pyranosylium ion-like TSs, and with catalysis driven solely by enzymatic H-bonding assistance. In chapter 6 I present the design and synthesis of a cyclopropyl inactivator candidate for β-d- glucuronidase and α-l-iduronidase which was tested for activity with human βglucuronidase and human α-l-iduronidase but found no inhibition against the natural substrate.