The affinity of 17β-estradiol (E2) for the estrogen receptor is weakened beyond the point of physiological relevance by the transfer of the sulfuryl moiety (-SO3) from PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 3′-hydroxyl of E2. The mechanism of this transfer reaction, catalyzed by estrogen sulfotransferase (EST), is investigated here in detail. The enzyme (a dimer of identical protomers) presents a clear example of half-sites reactivityonly one of the subunits of the dimer produces product during the catalytic cycle. This is the first example of half-sites reactivity in the sulfotransferase family. A burst of product, with an amplitude that corresponds to one-half of the available active sites, reveals that the mechanism is rate-limited by product release. The equilibrium constant governing interconversion of the substrate (EPAPSE 2) and product (EPAPE2S) central complexes was determined and is strongly biased toward product (Keq int ∼ 49). Slow product release allows the interconversion of the central complexes to approach equilibrium, with the result that Keq int becomes nearly linearly coupled to Km and contributes a factor of ∼30 to the steady-state affinity of the enzyme for substrate. Typical of its family, estrogen sulfotransferase is partially kcat-inhibited by its acceptor substrate, E2. This inhibition does not influence the burst kinetics and thus occurs after formation of the product central complex, a finding consistent with the slow escape of PAP from the nonreactive EPAPE2 complex.
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