All ETDs from UAB

Advisory Committee Chair

Charles N Falany

Advisory Committee Members

Stephen Barnes

Marry-Ann Bjornsti

Peter Prevelige, Jr

Yuhua Song

Document Type

Dissertation

Date of Award

2011

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Sulfation is an important Phase II drug metabolism reaction catalyzed by the cytosolic sulfotransferases (SULTs). SULT2A1 is a major SULT in liver and adrenal cortex that has been reported to sulfate a wide variety of substrates including bile acids, steroids, and drugs. The crystal structures of SULT2A1 suggest that PAPS binding causes a structural change. This study examines the kinetic changes in SULT2A1 caused by PAPS binding using computer modeling, enzyme kinetics, binding studies, and mammalian cells expressing SULT2A1. The data presented clearly demonstrate that the binding of PAPS changes the affinity of some substrates to SULT2A1 resulting in different apparent reaction mechanisms. With small substrates, such as dehydroepiandrosterone (DHEA), the binding of PAPS causes only a small change in substrate affinity to SULT2A1. For these substrates, either PAPS or acceptor may bind first resulting in a random Bi Bi reaction mechanism. With larger substrates, the binding of PAPS generated a conformational change in SULT2A1 that blocked the substrate from binding in a catalytic orientation. In these reactions, the substrate must bind before PAPS resulting in an ordered reaction mechanism. All human SULTs have been identified as homodimers. In SULT2A1, the addition of an amino-terminal maltose binding protein (MBP) tag disrupted dimerization. The MBP-SULT2A1 monomer was kinetically active and showed similar affinities for DHEA and PAPS as the SULT2A1 homodimer. However, the MBP-SULT2A1 monomer did not show substrate inhibition. Analysis of MBP-SULT2A1 and dimeric SULT2A1 demonstrated that substrate inhibition of DHEA was caused by the binding of DHEA at an inhibitory allosteric site. The inhibitory binding site was blocked or disrupted when PAPS bound to the homodimer. The molecular rearrangements observed in SULT2A1 upon PAPS binding were hypothesized to occur in other SULT isoforms. Modeling and enzyme kinetics demonstrated that similar as well as different structural changes occur in SULT1A1. These changes had a significant effect on the binding and kinetic properties of SULT1A1 The study presents novel insights into SULT2A1 and SULT1A1 structural, kinetic, and binding properties, and provides valuable insights for improving the in silico predictions of the in vivo sulfation of therapeutic drugs.

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