Homotetrameric MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis catalyses the NAD-dependent oxidation of MMSA (methylmalonate semialdehyde) and MSA (malonate semialdehyde) into PPCoA (propionyl-CoA) and acetyl-CoA respectively via a two-step mechanism. In the present study, a detailed mechanistic characterization of the MSDH-catalysed reaction has been carried out. The results suggest that NAD binding elicits a structural imprinting of the apoenzyme, which explains the marked lag-phase observed in the activity assay. The enzyme also exhibits a half-of-the-sites reactivity, with two subunits being active per tetramer. This result correlates well with the presence of two populations of catalytic Cys302 in both the apo- and holo-enzymes. Binding of NAD causes a decrease in reactivity of the two Cys302 residues belonging to the two active subunits and a pKapp shift from approx. 8.8 to 8.0. A study of the rate of acylation as a function of pH revealed a decrease in the pKapp of the two active Cys302 residues to approx. 5.5. Taken to-gether, these results support a sequential Cys302 activation process with a pKapp shift from approx. 8.8 in the apo-form to 8.0 in the binary complex and finally to approx. 5.5 in the ternary complex. The rate-limiting step is associated with the β-decarboxylation process which occurs on the thioacylenzyme intermediate after NADH release and before transthioesterification. These data also indicate that bicarbonate, the formation of which is enzyme-catalysed, is the end-product of the reaction.
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Research Article|
March 15 2006
Mechanistic characterization of the MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis
Claire Stines-Chaumeil;
Claire Stines-Chaumeil
1Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP, Université Henri Poincaré Nancy I, 54506 Vandoeuvre-lès-Nancy Cedex, France
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François Talfournier;
François Talfournier
1Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP, Université Henri Poincaré Nancy I, 54506 Vandoeuvre-lès-Nancy Cedex, France
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Guy Branlant
Guy Branlant
1
1Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP, Université Henri Poincaré Nancy I, 54506 Vandoeuvre-lès-Nancy Cedex, France
1To whom correspondence should be addressed (email guy.branlant@maem.uhp-nancy.fr).
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Publisher: Portland Press Ltd
Received:
September 16 2005
Revision Received:
December 06 2005
Accepted:
December 07 2005
Accepted Manuscript online:
December 07 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 395 (1): 107–115.
Article history
Received:
September 16 2005
Revision Received:
December 06 2005
Accepted:
December 07 2005
Accepted Manuscript online:
December 07 2005
Citation
Claire Stines-Chaumeil, François Talfournier, Guy Branlant; Mechanistic characterization of the MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis. Biochem J 1 April 2006; 395 (1): 107–115. doi: https://doi.org/10.1042/BJ20051525
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