The importance of Tyr-475 and Glu-506 in β-galactosidase from L. lactis ssp. lactis 7962

The secondary and tertiary structures of β-galactosidase from L. lactis ssp. lactis 7962 were designed using Nnpredict and Sybyl version 6.3. By using site-directed mutagenesis, the mutated enzymes, Tyr-475-Phe and Glu-506-Asp, were generated based on the structural modeling of L. lactis ssp. lactis 7962. The enzymes Tyr-475-Phe and Glu-506-Asp had <1% of the activity of the native enzyme with ONPG as substrate. The V_max values of the mutated enzymes were greatly reduced (1,800∼40,000-fold) compared with the value for the native β-galactosidase. However, the K_m values of Tyr-475-Phe and Glu-506-Asp with ONPG, PNPG, PNPF, and PNPA were not significantly different from those of the native enzyme. The results obtained support the suggestion that Tyr-475 and Glu-506 constitute very important parts of the catalytic machinery of the β-galactosidase.