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Abstract:
The induced-fit mechanism in Enterobacter cloacae MurA has been investigated by kinetic studies and X-ray crystallography. The antibiotic fosfomycin, an irreversible inhibitor of MurA, induced a structural change in UDP-N-acetylglucosamine (UDPGlcNAc)-liganded enzyme with a time dependence similar to that observed for the inactivation progress. The mechanism of action of fosfomycin on MurA appeared to be of the bimolecular type, the overall rate constants of inactivation and structural change being = 104 M-1 s-1 and = 85 M-1 s-1, respectively. Fosfomycin as well as the second MurA substrate, phosphoenolpyruvate (PEP), are known to interact with the side chain of Cys115. Like wild-type MurA, the catalytically inactive single-site mutant protein Cys115Ser structurally interacted with UDPGlcNAc in a rapidly reversible reaction. However, in contrast to wild-type enzyme, binding of PEP to mutant protein induced a rate-limited, biphasic structural change. Fosfomycin did not affect the structure of the mutant protein. The crystal structure of unliganded Cys115Ser MurA at 1.9 ? resolution revealed that the overall conformation of the loop comprising residues 112-121 is not influenced by the mutation. However, other than Cys115 in wild-type MurA, Ser115 exhibits two distinct side-chain conformations. A detailed view on the loop revealed the existence of an elaborate hydrogen-bonding network mainly supplied by water molecules, presumably stabilizing its conformation in the unliganded state. The comparison between the known crystal structures of MurA, together with the kinetic data obtained, suggest intermediate conformational states in the MurA reaction, in which the loop undergoes multiple structural changes upon ligand binding
