MUTATIONAL ANALYSIS OF THE CONSERVED CATIONIC RESIDUES OF BACILLUS-STEAROTHERMOPHILUS 6-PHOSPHOGLUCOSE ISOMERASE

The importance in catalysis of the conserved arginine (R207) and lysin e residues (K144, K294, K356, and K425) of 6-phosphoglucose isomerase from Bacillus stearothermophilus was assessed by site-directed mutagen esis and kinetic analysis. In general mutations had minor effects on t he K-m for fructose 6-phosphate. More dramatic effects were seen on k( cat). The R207A mutant had a five orders of magnitude decrease in k(ca t) relative to the wild-type enzyme. There was a significant recovery, by three orders of magnitude, in the k(cat) for the R207K mutant. The results suggest that the positive charge provided by R207 plays a cri tical role in the isomerization reaction. K425 was substituted with al anine, valine, phenylalanine, tryptoplian and aspartate. All mutant en zymes at position 425 had k(cat) decreased in the range of several-hun dred-fold. For the other mutants, K294A and K144A, the k(cat) values w ere 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 enzy me. The active-site location and the catalytic roles of K425 and K294 are supported further by the inhibitory effects of pyridoxal 5'-phosph ate on enzymatic activities. The data also confirm thr importance of K 425 and K144 anticipated by the affinity labeling studies of the corre sponding residues by pyridoxal 5'-phosphate in pig muscle phosphogluco se isomerase.