Date of Graduation

12-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor

Susanne Striegler

Committee Member

Roger Koeppe

Second Committee Member

Joshua Sakon

Third Committee Member

Suresh Kumar Thallapuranam

Keywords

beta-galactosidase, Glycoside Hydrolases, Inhibition, Transition State Analogs

Abstract

Glycoside hydrolases are ubiquitous and one of the most catalytically proficient enzymes known, and thus understanding their mechanisms are crucial. Most research has focused on the interaction of the glycon of substrates and their inhibitors within the active site of glycoside hydrolases. The inhibitors employed to probe these interactions generally had small aglycons (i.e. a hydrogen atom, amidines, small aliphatic groups, or benzyl groups). Here, the interactions of the aglycon with glycoside hydrolases are examined by probing the active sites with a library of 25 galactonoamidines. The studies described in this dissertation aim to increase the understanding of stabilization of the transition state by glycoside hydrolases, which allows for the acceleration of substrate hydrolysis by the enzymes up to 1017 over non-enzymatic hydrolysis. To understand this stabilization, the active sites of beta-galactosidases from Aspergillus oryzae, bovine liver, and Escherichia coli were evaluated using spectroscopic, molecular docking, and modeling analyses to determine transition state analogs (TSAs) and how the TSAs interact within the active site of glycoside hydrolases.

The probing with the galactonoamidine library revealed hydrophobic interactions, pi-pi interactions, and CH-pi interactions within the active sites to varying extent. Further, three TSAs were found for the hydrolysis of substrates by beta-galactosidase (A. oryzae), and two TSAs for the beta-galactosidases from bovine liver and E. coli. Upon TSA binding to the three beta-galactosidases, conformational changes occurred to stabilize the galactonoamidines within the active sites, which did not occur when fortuitous binders interacted with the enzyme. The conformational changes within the active sites of beta-galactosidases from bovine liver and E. coli closes off the active site via a loop movement resulting in a substantially higher binding affinity than those observed with beta-galactosidase (A. oryzae). A subsequent evaluation of galactonoamidine specificity in the presence of other proteins revealed an increase of inhibitory activity two orders of magnitude more than a purified beta-galactosidase (E. coli).

Comments

The supplemental files are data files referenced within this dissertation.

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