Exploring the nucleotide-binding site in proteins by affinity labeling and site-directed mutagenesis

Affinity labeling with nucleoside polyphosphopyridoxals, especially those with 3 or 4 phosphate groups, was effective for identifying the lysyl residue(s) located at or near the binding site for ATP, GTP, UDP-Glc, or ADP-Glc in various proteins. Furthermore, kinetic analysis of the mutant enzymes, in which the labeled lysyl residue was replaced with another amino acid by site-directed mutagenesis, provided evidence of its functional role. Affinity labeling of the mutant enzymes was useful for further identification of the hidden lysyl residue, which is unreactive in the wild-type enzyme but catalytically important. Comparison of the results of affinity labeling with different substrate analogues provided the information on the location of the labeled lysyl residue around the bound substrate. The affinity labeling reflected structural features of proteins, including their conformational flexibility.