Mutational analysis of the conserved cationic residues of Bacillus stearothermophilus 6-phosphoglucose isomerase.
The importance in catalysis of the conserved arginine (R207) and lysine residues (K144, K294, K356, and K425) of 6-phosphoglucose isomerase from Bacillus stearothermophilus was assessed by site-directed mutagenesis and kinetic analysis. In general mutations had minor effects on the Km for fructose 6-phosphate. More dramatic effects were seen on kcat. The R207A mutant had a five orders of magnitude decrease in kcat relative to the wild-type enzyme. There was a significant recovery, by three orders of magnitude, in the kcat for the R207K mutant. The results suggest that the positive charge provided by R207 plays a critical role in the isomerization reaction. K425 was substituted with alanine, valine, phenylalanine, tryptophan and aspartate. All mutant enzymes at position 425 had kcat decreased in the range of several-hundred-fold. For the other mutants, K294A and K144A, the kcat values were 3.5% and 27% of the wild-type enzyme, respectively. No effects on catalysis were observed for the K356A mutant. The results suggest that R207, K144, K294, and K425 are located in the active site of the enzyme. The active-site location and the catalytic roles of K425 and K294 are supported further by the inhibitory effects of pyridoxal 5'-phosphate on enzymatic activities. The data also confirm the importance of K425 and K144 anticipated by the affinity labeling studies of the corresponding residues by pyridoxal 5'-phosphate in pig muscle phosphoglucose isomerase.
Meng
M
M
Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, Taichung, Taiwan. mhmeng@dragon.nchu.edu.tw
Chen
Y T
YT
Hsiao
Y Y
YY
Itoh
Y
Y
Bagdasarian
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002412:Cations; D002942:Circular Dichroism; D017931:DNA Primers; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005956:Glucose-6-Phosphate Isomerase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid
D002412:Cations; D017931:DNA Primers; D005956:Glucose-6-Phosphate Isomerase