The B-12-binding subunit of glutamate mutase from Clostridium tetanomorphum traps the nucleotide moiety of coenzyme B-12

Glutamate mutase from Clostridium tetanomorphum binds coenzyme B-12 in a ba se-off/His-on form, in which the nitrogenous Ligand of the B-12-nucleotide function is displaced from cobalt by a conserved histidine. The effect of b inding the B-12-nucleotide moiety to MutS, the B-12-binding subunit of glut amate mutase, was investigated using NMR spectroscopic methods. Binding of the B-12-nucleotide to MutS was determined to occur with K-d = 5.6(+/-0.7) mM and to be accompanied by a specific conformational change in the protein . The nucleotide binding cleft of the apoprotein, which is formed by a dyna mic segment with propensity for partial a-helical conformation (the "nascen t" alpha -helix), becomes completely structured upon binding of the B-12-nu cleotide, with formation of helix alpha1. In contrast, the segment containi ng the conserved residues of the B-12-binding Asp-x-His-x-x-Gly motif remai ns highly dynamic in the protein/ B-12-nucleotide complex. From relaxation studies, the time constant tau, which characterizes the time scale for the formation of helix alpha1, was estimated to be about 30 mus N-15 and was th e same in both, ape-protein and nucleotide-bound protein. Thus, the binding of the B-12-nucleotide moiety does not significantly alter the kinetics of helix formation, but only shifts the equilibrium towards the structured fo ld. These results indicate MutS to be structured in such a way, as to be ab le to trap the nucleotide segment of the base-off form of coenzyme B-12 and provide, accordingly, the first structural clues as to how the process of B-12-binding occurs. (C) 2001 Academic Press.