Role of Met-542 as a guide for the conformational changes of Phe-601 that occur during the reaction of β-galactosidase (Escherichia coli)

The Met-542 residue of β-galactosidase is important for the enzyme’s activity because it acts as a guide for the movement of the benzyl side chain of Phe-601 between two stable positions. This movement occurs in concert with an important conformational change (open vs. closed) of an active site loop (residues 794–803). Phe-601 and Arg-599, which interact with each other via the π electrons of Phe-601 and the guanidium cation of Arg-599, move out of their normal positions and become disordered when Met-542 is replaced by an Ala residue because of the loss of the guide. Since the backbone carbonyl of Phe-601 is a ligand for Na + , the Na + also moves out of its normal position and becomes disordered; the Na + binds about 120 times more poorly. In turn, two other Na + ligands, Asn-604 and Asp-201, become disordered. A substrate analog (IPTG) restored Arg-599, Phe-601, and Na + to their normal open-loop positions, whereas a transition state analog (d-galactonolactone) restored them to their normal closed-loop positions. These compounds also restored order to Phe-601, Asn-604, Asp-201, and Na + . Binding energy was, however, necessary to restore structure and order. The K s values of oNPG and pNPG and the competitive K i values of substrate analogs were 90–250 times higher than with native enzyme, whereas the competitive K i values of transition state analogs were ~3.5–10 times higher. Because of this, the E•S energy level is raised more than the E•transition state energy level and less activation energy is needed for galactosylation. The galactosylation rates (k 2 ) of M542A–β-galactosidase therefore increase. However, the rate of degalactosylation (k 3 ) decreased because the E•transition state complex is less stable.