Functional analysis of conserved histidines in ADP-glucose pyrophosphorylase from Escherichia coli

Two absolutely conserved histidines and a third highly conserved histidine are noted in 11 bacterial and plant ADP-glucose pyrophosphorylases. These histidines were individually mutagenized in the E. coli enzyme to glutamine in order to determine their function. Glutamine mutations at residues 143 and 156 produced functional enzymes in cell extracts with slightly lower than wild-type specific catalytic activities and with same heat stability characteristics of the wild-type enzyme. Substitution of residue 83 with glutamine however produced an enzyme having decreased thermal stability. Additional mutageneses at residue 83 with asparagine, arginine, or aspartate gave rise to enzymes having a progressively decreasing trend in thermal stability. These mutants are more susceptible to proteolysis than wild-type enzyme. Kinetic analysis of H83Q and H83N indicates that histidine 83 is not involved in the catalytic mechanism or in substrate binding but possibly in maintenance of the active catalytic structure.