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 base-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 binding the B(12)-nucleotide moiety to MutS, the B(12)-binding subunit of glutamate 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 apo-protein, which is formed by a dynamic segment with propensity for partial alpha-helical conformation (the "nascent" alpha-helix), becomes completely structured upon binding of the B(12)-nucleotide, with formation of helix alpha1. In contrast, the segment containing the conserved residues of the B(12)-binding Asp-x-His-x-x-Gly motif remains 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 micros (15)N and was the same in both, apo-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 fold. These results indicate MutS to be structured in such a way, as to be able 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.

Protein-coenzyme interactions in adenosylcobalamin-dependent glutamate mutase.

Glutamate mutase catalyses an unusual isomerization involving free-radical intermediates that are generated by homolysis of the cobalt-carbon bond of the coenzyme adenosylcobalamin (coenzyme B(12)). A variety of techniques have been used to examine the interaction between the protein and adenosylcobalamin, and between the protein and the products of coenzyme homolysis, cob(II)alamin and 5'-deoxyadenosine. These include equilibrium gel filtration, isothermal titration calorimetry, and resonance Raman, UV-visible and EPR spectroscopies. The thermodynamics of adenosylcobalamin binding to the protein have been examined and appear to be entirely entropy-driven, with DeltaS=109 J.mol(-1).K(-1). The cobalt-carbon bond stretching frequency is unchanged upon coenzyme binding to the protein, arguing against a ground-state destabilization of the cobalt-carbon bond of adenosylcobalamin by the protein. However, reconstitution of the enzyme with cob(II)alamin and 5'-deoxyadenosine, the two stable intermediates formed subsequent to homolysis, results in the blue-shifting of two of the bands comprising the UV-visible spectrum of the corrin ring. The most plausible interpretation of this result is that an interaction between the protein, 5'-deoxyadenosine and cob(II)alamin introduces a distortion into the ring corrin that perturbs its electronic properties.