We have investigated the involvement of metal ions and conformational changes in the elimination reaction catalysed by type II dehydroquinase from Aspergillus nidulans. Mechanistic comparisons between dehydroquinases and aldolases raised the possibility that, by analogy with type II aldolases, type II dehydroquinases may require bivalent metal ions for activity. This hypothesis was tested by a combination of metal analysis, effects of metal chelators and denaturation/renaturation experiments, all of which failed to show any evidence that type II dehydroquinases are metal-dependent dehydratases. Analysis of native and refolded enzyme by electron microscopy showed that the dodecameric type II enzyme from A. nidulans adopts a ring-like structure similar to that of glutamine synthase, suggesting an arrangement of two hexameric rings stacked on top of one another. Evidence for a ligand-induced conformational change came from both chemical modification and proteolysis experiments. Inactivation data with the arginine-specific reagent phenylglyoxal indicated that, at pH 7.5, two arginine residues are modified: one modification displays affinity-labelling kinetics and has a 1:1 stoichiometry, while the other displays simple bimolecular kinetics and a stoichiometry of 2:1. The labelling at the affinity site is markedly enhanced by the addition of ligand, implying that this active-site residue is further exposed to modification by phenylglyoxal as a result of a ligand-induced conformational change. A combination of proteolysis and electrospray MS experiments identified the site of affinity labelling as Arg-19. The highly conserved N-terminal region encompassing Arg-19 of type II dehydroquinase was found to be particularly susceptible to proteolytic cleavage. Limited digestion with proteinase K inactivates the enzyme, although the type II oligomeric structure is retained, and ligand binding partially protects against this inactivation.
Conformational changes and the role of metals in the mechanism of type II dehydroquinase from Aspergillus nidulans
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Joanna R BOTTOMLEY, Alastair R. HAWKINS, Colin KLEANTHOUS; Conformational changes and the role of metals in the mechanism of type II dehydroquinase from Aspergillus nidulans. Biochem J 1 October 1996; 319 (1): 269–278. doi: https://doi.org/10.1042/bj3190269
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