[68] Pyruvate dehydrogenase complex from Bacillus

Publisher Summary The pyruvate dehydrogenase complex is a multienzyme complex catalyzing the oxidative decarboxylation of pyruvate to acetyl coenzyme (CoA). This chapter describes the assay method and properties of pyruvate dehydrogenase complex isolated from Bacillus . The structural organization of the bacillar complex differs substantially from that of E. coli and of other prokaryotes. Bacillus spp . is gram-positive and the enzymes of some species are thermostable. The purification of the enzyme complexes of B. subtilis and B. stearothermophilus by using affinity chromatography and some properties of the complexes are also described in the chapter. The enzyme complex is assayed spectrophotometrically by following the keto acid-dependent reduction of either 3-acetylpyridine nicotinamide adenine dinucleotide (NAD + ) or NAD + at 366 nm or 340 nm. NAD + is preferred in the assay because it is cheaper than 3-acetylpyridine NAD + . In crude extracts, 3-acetylpyridine NAD + is preferred because of the interference of nicotinamide adenine dinucleotide dehydrogenase (NADH) oxidase in the NAD + assay. The recovery of the pyruvate dehydrogenase complex on ethanol–Sepharose 2B starting with crude extract is generally high.