Role of the Axial Ligand in Catalysis

For glutamate mutase, site specific mutagenesis of the conserved histidine and aspartate residues has been performed (Chen and Marsh, 1997). Mutation of either the histidine or the aspartate results in a dramatic 

FIGURE 18. Low frequency resonance Raman spectra of AdoCbl in free solution and bound to methylmalonyl-CoA mutase. Inset are spectra obtained with [5 - C]-AdoCbl that identify the CooC stretch and die 6-ribose vibrations. It is evident that neidier of these vibrational modes in changed greatly when the coenzyme is bound by the protein. Reprinted with permission from Dong et ah, 1998, copyright 1998 American Chemical Society. 

These experiments serve to demonstrate the importance of these residues in catalysis, but their exact role is still not fully understood. Since the mutant enzymes no longer accumulate Cbl(ll) during steady state turnover, it is reasonable to assume that the mutations affect the ability of the enzyme to break the CooC bond. However, the decrease in although large, represents no more than lO of the lO -fold increase in the rate of AdoCbl homolysis required to explain the observed rate of turnover (Hay and Finke, 1987 Marsh and Ballou, 1998). This observatiou, aud the fact that enzymes such as diol dehydrase bind AdoCbl with the coenzyme nucleotide tail still coordinated to cobalt (Yamanishi et al., 1998b), suggest that the histidine-aspartate pair exerts a relatively small effect on the reactivity of the coenzyme and serves only to fine-tune catalysis. 

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