Mechanistic characterization of the MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis.
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 b-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.
Stines-Chaumeil
Claire
C
Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP, Université Henri Poincaré Nancy I, 54506 Vandoeuvre-lès-Nancy Cedex, France.
Talfournier
François
F
Branlant
Guy
G
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000737:chemistry; D001051:Apoenzymes / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology*; D001639:Bicarbonates; D002384:Catalysis; D003545:Cysteine / Q000378:metabolism; D003653:Decarboxylation; D004220:Disulfides / Q000737:chemistry; D020035:Holoenzymes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000737:chemistry; D007700:Kinetics; D050646:Methylmalonate-Semialdehyde Dehydrogenase (Acylating) / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D013997:Time Factors
D001051:Apoenzymes; D001639:Bicarbonates; D004220:Disulfides; D020035:Holoenzymes; D009243:NAD; C001105:2,2'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; D050646:Methylmalonate-Semialdehyde Dehydrogenase (Acylating); D003545:Cysteine; D007460:Iodoacetamide