HUMAN GLUCOSE-6-PHOSPHATE-DEHYDROGENASE - LYSINE-205 IS DISPENSABLE FOR SUBSTRATE-BINDING BUT ESSENTIAL FOR CATALYSIS

By site-directed mutagenesis of the cloned human glucose-6-phosphate d ehydrogenase cDNA, lysine 205 (the residue that after reacting with py ridoxal-5'-phosphate renders inactive enzyme) was mutated to threonine (K205T) to remove the amino group, or to arginine (K205R) to displace the position of the amino group, in order to analyze the role of its nucleophilic group in position epsilon. Compared to the wild-type enzy me, the K205T and K205R mutants retain a specific activity of 2.6 and 11.4%, respectively; their catalytic specificity (K-cat/K-m) is drasti cally decreased, whereas the K-m values for both substrates are only s lightly increased, These findings in the light of the 3D structure of G6PD suggest that the epsilon-amino group of lysine 205 can favour a h ydrogen bond within the active pocket essential for catalysis.