Adenosylcobalamin-dependent glutamate mutase: examination of substrate and coenzyme binding in an engineered fusion protein possessing simplified subunit structure and kinetic properties.
Glutamate mutase is comprised of two weakly associating subunits; E and S, that combine to form the coenzyme binding site. The active holoenzyme assembles in a kinetically complex process in which both the stoichiometry and apparent Kd for adenosylcobalamin (AdoCbl) are dependent upon the relative concentrations of the two subunits, as is the enzyme's specific activity. To facilitate mechanistic and structural studies on this enzyme we have genetically fused the S subunit to the C-terminus of the E subunit through an 11 amino acid (Gly-Gln)5-Gly linker segment. This protein, GlmES, binds AdoCbl stoichiometrically and neither the affinity for AdoCbl nor the turnover number depends upon protein concentration. The kcat and Km for both substrate and coenzyme, together with the deuterium isotope effects on Vmax and Vmax/Km, have been determined for the GlmES-catalyzed reaction proceeding in both directions. Compared with wild type, the affinity for AdoCbl is unchanged, but for the conversion of L-glutamate to (2S,3S)-3-methylaspartate both kcat and Km for L-glutamate are decreased by about a third and the isotope effects are reduced, suggesting product release to be more rate-limiting. To test hypotheses concerning the activation of the coenzyme, we examined the binding of adenosylcobalamin, methylcobalamin, and cob(II)alamin to the enzyme. Each of these is bound with essentially the same affinity (2 microM), suggesting that, contrary to expectations, interactions between the protein and the adenosyl moiety do not serve to weaken the cobalt-carbon bond in the ground state.
Chen
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Department of Chemistry, University of Michigan, Ann Arbor 48109-1055, USA.
Marsh
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D001051:Apoenzymes / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology / Q000235:genetics; D003038:Cobamides / Q000378:metabolism*; D003903:Deuterium; D004926:Escherichia coli / Q000235:genetics; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001051:Apoenzymes; D003038:Cobamides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D003903:Deuterium; D019751:Intramolecular Transferases; C021570:methylaspartate mutase; C000913:cobamamide