Mechanistic characterization of the MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis

• Published in: Biochemical journal 2006-04, Vol.395 (1), p.107-115
• Main Authors: Stines-Chaumeil, Claire, Talfournier, François, Branlant, Guy
• Format: Article
• Language: English
• Subjects: 2,2'-Dipyridyl - analogs & derivatives; 2,2'-Dipyridyl - chemistry; Apoenzymes - metabolism; Bacillus subtilis; Bacillus subtilis - enzymology; Bicarbonates; Catalysis; Cysteine - metabolism; Decarboxylation; Disulfides - chemistry; Holoenzymes - metabolism; Hydrogen-Ion Concentration; Iodoacetamide - chemistry; Kinetics; Methylmalonate-Semialdehyde Dehydrogenase (Acylating) - metabolism; Mutation - genetics; NAD - metabolism; Oxidation-Reduction; Protein Binding; Time Factors
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj20051525

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.