IDENTIFICATION OF AMINO-ACID-RESIDUES CRITICAL FOR CATALYSIS AND COSUBSTRATE BINDING IN THE FLAVONOL 3-SULFOTRANSFERASE

The comparison of the deduced amino acid sequences of plant and animal sulfotransferases (ST) has allowed the identification of four well co nserved regions, and previous experimental evidence suggested that reg ions I and IV might be involved in the binding of the cosubstrate, 3'- phosphoadenosine 5'-phosphosulfate (PAPS), Moreover, region IV is homo logous to the glycine-rich phosphate binding loop (P-loop) motif known to be involved in nucleotide phosphate binding in several protein fam ilies. In this study, the function of amino acid residues within these two regions was investigated by site directed mutagenesis of the plan t flavonol 3-ST. In region I, our results identify Lys(59) as critical for catalysis, since replacement of this residue with alanine re suit ed in a 300-fold decrease in specific activity, while a 15-fold reduct ion was observed after the conservative replacement with arginine. Pho toaffinity labeling of K59R and K59A with [S-35]PAPS revealed that Lys (59) is not required for cosubstrate binding, However, the K59A mutant had a reduced affinity for 3'-phosphoadenosine 5'-phosphate (PAP)-aga rose, suggesting that Lys(59) may participate in the stabilization of an intermediate during the reaction, In region TV, all substitutions o f Arg(276) resulted in a marked decrease in specific activity. Conserv ative and unconservative replacements of Arg(276) resulted in weak pho toaffinity labeling with [S-35]PAPS and the R276A/T73A and R276E enzym es displayed reduced affinities for PAP-agarose, suggesting that the A rg(276) side chain is required to bind the cosubstrate, The analysis o f the kinetic constants of mutant enzymes at residues Lys(277), Gly(28 1), and Lys(284) allowed to confirm that region TV is involved in cosu bstrate binding.