F. Marsolais et L. Varin, IDENTIFICATION OF AMINO-ACID-RESIDUES CRITICAL FOR CATALYSIS AND COSUBSTRATE BINDING IN THE FLAVONOL 3-SULFOTRANSFERASE, The Journal of biological chemistry, 270(51), 1995, pp. 30458-30463
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.