The human estrogen sulfotransferase: a half-site reactive enzyme.

The affinity of 17beta-estradiol (E(2)) for the estrogen receptor is weakened beyond the point of physiological relevance by the transfer of the sulfuryl moiety (-SO(3)) from PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 3'-hydroxyl of E(2). 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 reactivity--only 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 (E.PAPS.E(2)) and product (E.PAP.E(2)S) central complexes was determined and is strongly biased toward product (K(eq int) approximately 49). Slow product release allows the interconversion of the central complexes to approach equilibrium, with the result that K(eq int) becomes nearly linearly coupled to K(m) and contributes a factor of approximately 30 to the steady-state affinity of the enzyme for substrate. Typical of its family, estrogen sulfotransferase is partially k(cat)-inhibited by its acceptor substrate, E(2). 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 E.PAP.E(2) complex.