All ETDs from UAB

Advisory Committee Chair

Charles N Falany

Advisory Committee Members

Mahmoud H El Kouni

Stephen Barnes

Teresa W Wilborn

Peter E Prevelige

Document Type

Dissertation

Date of Award

2016

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The human cytosolic sulfotransferases (hSULTs) are a fourteen-member family of phase II drug-metabolizing enzymes that catalyze the transfer of a sulfonate moiety from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to a recipient substrate. SULT-mediated sulfation serves to deactivate physiological hormones and detoxify xenobiotics. SULT1B1 is primarily resident to the gastrointestinal tract, liver, and possibly peripheral white blood cells (WBCs), whereby it performs its supposed physiological role. The iso-form has the capacity to sulfate thyroid hormones, small phenols, and polyaromatic hy-drocarbons resulting in their inactivation, detoxification, and bioactivation/detoxification, respectively. Immunohistochemistry was used to show hSULT1B1 protein is present in periph-eral lymphocytes and neutrophils. Further, a novel hSULT1B1 single nucleotide poly-morphism (SNP) was identified which resulted in the substitution of Leu145 with a Val. The SNP was specific to Sub-Saharan African humans with an allelic frequency of ap-proximately 10%. The location of the -L145V substitution on hSULT1B1’s structure prompted the investigation of its biochemical characteristics after recombinant expression and purification. The -L145V isoform was deficient in its maximal turnover rate with re-spect to PAPS. The -L145V isoform also exhibited increased susceptibility to substrate inhibition, attributed to its favorable Kd for the reaction byproduct, PAP. The altered ac-tivity of -L145V prompted an association study of the SNP with colorectal and prostate cancer. No significant correlation between the genotype and diseases were observed. Each SULT is a dimer interfacing along a conserved C-terminal KxxxTVxxxE motif. Recent evidence points toward a mechanistic role where a single subunit of the SULT dimer is active at any given time, termed half-site reactivity (HSR). HSR requires communication of the subunits through the dimerization domain. Molecular dynamic simulation results showed PAP(S) binding was capable of inducing this cross-talk via its interaction with a conserved residue, Arg258. This observation was tested in vitro using monomeric and self-dimerizing hSULT1B1 subunits, generated by directed mutation of the dimerization domain. Arg258 and His109 (catalytic base) were mutated in a single dimer subunit to test the dependence of one subunit’s activity on its partner’s activity. No significant intersubunit dependence was detected with respect to the neighboring subu-nit’s activity, suggesting hSULT1B1 is not a half-site reactive isoform.

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