Substitutions for Cys-472 and His-509 at the active site of β-galactosidase from Lactococcus lactis ssp. lactis 7962 cause large decreases in enzyme activity

Structural modeling of β-galactosidase from L. lactis ssp. lactis 7962 has shown that the residues Cys-472 and His-509 are located in the wall of the active-site cavity. To examine the functions of Cys-472 and His-509, we generated five site-specific mutants: Cys-472-Ser, Cys-472-Thr, Cys-472-Met, His-509-Asn, and His-509-Phe. β-Galactosidase substituted at Cys-472 with Met or His-509 with Phe had <3% of the activity of the native enzyme when assayed using ONPG as substrate. The other mutants Cys-472-Ser, Cys-472-Thr, and His-509-Asn had ca. 10-15% of the native enzyme activity. The V max values of the five mutated enzymes were lower (60-7,000-fold) than that of native enzyme. These results show that the catalytic ability of β-galactosidase is significantly affected by mutations at Cys-472 or His-509.