All ETDs from UAB

Advisory Committee Chair

Charles N Falany

Advisory Committee Members

Karina Yoon

Stephen Barnes

Peter E Prevelige, Jr

Harald W Sontheimer

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Human cytosolic sulfotransferases (SULTs) are a family of Phase II conjugating enzymes that facilitate the transfer of a sulfonate moiety from 3’phosphoadenosine 5’phosphosulfate (PAPS) to hydroxyl or amine groups of acceptor substrates. SULT1A1, located in many tissues throughout the human body including the liver, is important in the metabolism of many endogenous, exogenous, xenobiotic, and drug compounds. The majority of substrates for SULT1A1 are small neutral phenols including 1-naphthol and acetaminophen; however, SULT1A1 sulfates larger compounds including 17β-estradiol (E2) and raloxifene. SULT1A1 conjugates E2 with a Km of 2.3 µM and the structure of E2 is almost identical to the estrogenic component used in oral contraceptives, 17α-ethinylestradiol (EE2). The similarities in structure led to molecular modeling studies of both E2 and EE2 with SULT1A1. The result of the EE2 studies indicated that EE2 may potently inhibit SULT1A1 sulfation activity with PAPS present. The kinetic properties of SULT1A1 for EE2 showed the Km was lower than that of E2, EE2-sulfate was not detectable below 100 nM, and EE2 preferentially binds to the SULT1A1•PAP complex at concentrations below sulfation (Kd = 5 nM). EE2 was determined to potently inhibit SULT1A1 sulfation activity for known substrates with a K¬i of 9-20 nM and potent inhibition was detected in MCF-7 cells expressing SULT1A1 with an IC50¬ of 20 nM. Molecular modeling indicated interactions with Tyr169 and Ile89 may be necessary for the inhibitory conformation of EE2. Mutational analysis was performed and Ile89 has a major role in the inhibitory conformation of EE2 within SULT1A1 as mutation of Ile89 to Ala89 resulted in complete loss of potent inhibition of SULT1A1 by EE2. Tyr169 has a smaller role in the inhibitory conformation of EE2; however, the conservative mutation of SULT1A1-Tyr169Phe resulted in an increase in K¬i of EE2 and altered the binding affinity of E2 for SULT1A1-Tyr169Phe. A pilot human study was performed to determine in vivo interactions between EE2, acetaminophen, and SULT1A1. Co-administration of EE2 and acetaminophen lowered the AUC(0-4) of acetaminophen compared to acetaminophen alone. Acetaminophen-sulfate needs to be determined directly to know the effect of EE2 on SULT1A1 activity in vivo.

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