Add time:09/02/2019         Source:sciencedirect.com

Phenylglyoxal, a chemical modifying agent for arginine residues, produced rapid inactivation of a rat liver phenol sulfotransferase (3-phosphoadenylylsulfate:phenol sulfotransferase, EC 2.8.2.1). Enzyme inactivation was accompanied by incorporation of 1.5 mol [7−14C]phenylglyoxal per mol enzyme. 3′-Phosphoadenosine 5′-phosphosulfate (PAPS), the sulfate donor, prevented inactivation and decreased [7−14C]phenylglyoxal incorporation to 0.78 mol/mol enzyme. The sulfhydryl-modifying agent, N-ethylmaleimide, also caused rapid inactivation of phenol sulfotransferase with concomitant incorporation of 2.35 mol N-[3H]ethylmaleimide per mol enzyme. These results suggest a possible role for arginine residues as anionic recognition sites for the sulfate donor PAPS, and indicate the presence of essential sulfhydryl residues on phenol sulfotransferase. Ribonucleotide dialdehydes (ATPDA, ADPDA, AMPDA, APSDA), but not the corresponding 2′, 3′-acyclic nucleotides (ATPDO, ADPDO AMPDO, APSDO), produced rapid and irreversible inactivation of phenol sulfotransferase. These ribonucleotide dialdehydes appear to modify the active site of the enzyme, since inclusion of the sulfate donor, PAPS, or the product, adenosine 3′, 5′-bisphosphate (PAP), in the incubation mixture prevented loss of enzyme activity. In contrast, the sulfate acceptor, p-nitrophenol, did not show similar protective effects. Kinetic studies indicated that the ribonucleotide dialdehydes inactivated the enzyme via a unimolecular reaction within a dissociable enzyme-inhibitor complex rather than via a nonspecific bimolecular process. Radioactively labeled ribonucleotide dialdehydes (e.g., [2, 8−3H]ATP) were incorporated into protein concomitant with loss of enzyme activity. The incorporated ligand could be removed by dialysis in phosphate or Tris buffer. The protein-ligand complex could be stabilized to dialysis by pretreatment with sodium borohydride. The results of these studies suggest that ribonucleotide dialdehydes are affinity labeling reagents for phenol sulfotransferase, causing enzyme inactivation by the possible formation of a Schiff base adduct with an active-site lysine residue.

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