Interaction of the lacZ β-galactosidase of Escherichia coli with some β-d-galactopyranoside competitive inhibitors

Abstract

1. The location of the bivalent metal cation with respect to bound competitive inhibitors in Escherichia coli (!lacZ) β-galactosidase was investigated by proton magnetic resonance. 2. Replacement of Mg2+ by Mn2+ enhances both longitudinal and transverse relaxation of the methyl groups of the β-d-galactopyranosyltrimethylammonium ion, and of methyl 1-thio-β-d-galactopyranoside; linewidths are narrowed by increasing temperature. 3. The Mn2+ ion is located 8–9Å (0.8–0.9nm) from the centroid of the trimethylammonium group and 9Å (0.9nm) from the average position of the methylthio protons. 4. The effective charge at the active site was probed by measurement of competitive inhibition constants (Kio and Ki+ respectively) for the isosteric ligands, β-d-galactopyranosylbenzene and the β-d-galactopyranosylpyridinium ion. 5. The ratio of inhibition constants (Q=Ki+/Kio) obtained with 2-(β-d-galactopyranosyl)–naphthalene and the β-d-galactopyranosylisoquinolinium ion at pH7 with Mg2+–enzyme was identical, within experimental error, with that obtained with the monocyclic compounds. 6. The variation of Q for Mg2+–enzyme can be described by Q=0.1(1+[H+]/4.17×10−10)/1+[H+]/10−8). 7. This, in the theoretical form for a single ionizable group, is ascribed to the ionization of the phenolic hydroxy group of tyrosine-501. 8. The variation of Q for Mg2+-free enzyme is complex, probably because of deprotonation of the groups normally attached to Mg2+ as well as tyrosine-501.