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

Abstract

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 Ksvalues of oNPG and pNPG and the competitive Kivalues of substrate analogs were 90–250 times higher than with native enzyme, whereas the competitive Kivalues 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 (k2) of M542A–β-galactosidase therefore increase. However, the rate of degalactosylation (k3) decreased because the E•transition state complex is less stable.