Inhibition of phenol sulfotransferase by pyridoxal phosphate

Abstract

The biologically abundant cofactor, pyridoxal-5-phosphate (PLP), is a potent inhibitor of bovine phenol (aryl) sulfotransferase (PST). Preincubation of purified enzyme with as little as 1 μM PLP decreased PST activity by 50%. Excess 2-naphthol protected PST from inactivation by PLP, whereas 2-naphthyl sulfate and PAPS were not protective. Although PLP inhibition was apparently competitive with 2-naphthol, a steady-state kinetic K i value could not be measured due to non-linear Lineweaver-Burk plots in the presence of the inhibitor. Kinetic progress curves revealed that this was due to progressive loss of activity during catalysis. The kinetics of inactivation of PST by PLP were pseudofirst-order and exhibited saturation. The derived K I value for the binding of PLP to PST in the initial reversible step was 23 μM, with a maximal rate of inactivation of 0.077 min −1. Absorbance spectra of the PST/PLP complex indicated the formation of a Schiff base conjugate, and this is consistent with decreased electrophoretic mobility of the protein-PLP adduct in the presence of dodecyl sulfate only after reduction with borohydride. These results point to the possible regulation of an important detoxification enzyme by a ubiquitous cofactor.