Pyruvate Dehydrogenase Complex from Baker's Yeast. 1. Purification and Some Kinetic and Regulatory Properites

Abstract

Pyruvate dehydrogenase complex, for the first time, was highly purified from commercial baker's yeast (Saccharomyces cerevisiae). Proteolytic degradation was prevented by the inclusion of the protease inhibitors pepstatin A, leupeptin, and phenylmethanesulfonyl fluoride during the enzyme purification. The yield from 1 kg of pressed yeast was about 15-20mg enzyme with a spccific activity of 17-30U/mg. Most of the kinetic and regulatory properties of the yeast enzyme were found similar to those of the mammalian mitochondria1 pyruvate dehydrogenase complexes except that K, for pyruvate, when assayed at the pH optimum, was much higher than in the mammalian complexes and resembled the values reported for the complexes of gram-negative bacteria. Furthermore, neither in yeast homogenates nor in the isolated yeast pyruvate dehydrogenase complex, was any evidence found for regulation by interconversion (phosphorylation-dephosphorylation) as occurs in mammals, plants, and Neurospora crassuThe pyruvate dehydrogenase multienzyme complex occupies a central metabolic position connecting the mainstream carbohydrate metabolism with energy generation via the Krebs cycle. Consequently, the complex is an important point of regulation in many prokaryotic as well as eukaryotic organisms (review [I -41).The situation is somewhat complicatcd in facultative anaerobes such as Succharomyws cwevisiae. Knowledge about the properties and rcgulation of yeast pyruvate dehydrogcnase complex is incomplete. Nevertheless, the complex has been inferred to account for the major part of respiration in yeast growing aerobically whereas the main pathway of pyruvate catabolism under anaerobic conditions is thought to be decarboxylation catalyzed by pyruvate decarboxylase [5,6]. The presence ofpyruvate dehydrogenase complex however has also been demonstrated in anaerobically-grown yeast (7 -91.Pyruvate dchydrogenase complexes have been purified from many organisms including bacteria [lo-151, fungi [I 61, invertebrates [17], higher plants [18,19], birds [20,21], and mammals [22-251. Isolation of the complex from the yeasts Hanseizula miso [26] and Succharomyces carlsbergensis [9] has been reported, but these preparations were far from satisfactory with regard to the yield and stability of thc enzyme, mainly because of rapid protcolytic inactivation of the enzyme complex [9]. A similar high protease sensitivity has been detected in the pyruvate dehydrogenase complexes from Escherichia c d i [27,28] and mammalian liver [25,29]

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621

“…3). The affinity constant for pyruvate was about 0.3 raM, which is near the K m value that has been reported for the pyruvate dehydrogenase complex [14] and the K m value cf pyruvate transport by mitochondria from various sources [18]. These results make clear that, if the in vivo rates of pyruvate or malate oxidation by the two yeasts would be different, this could only result from differences in the intracelhilar concentration of these substrates.…”

“…These concentrations are saturating and non-inhibitory to the pyruvate dehydrogenase complex, as was established from oxygen consumption experiments with mitochondria. Pyruvate decarboxylase activity, however, is enhanced at 15 mM pyruvate, due to the low affinity of the enzyme for its substrate [10,14]. From the difference in 14CO2 production at the two pyruvate concentrations the contribution of contaminating pyruvate decarboxylase may be calculated.…”

The pyruvate dehydrogenase complex of Saccharomyces cerevisiae is regulated by phosphorylation

“…Previous attempts to detect yeast PDC kinase [7,8] have been unsuccessful, probably because PDC has been purified from bakers' yeast strains, as it now appears that yeast must be cultured under carefully defined conditions to detect phosphorylation of the complex. Moreover, in our hands, it has proved difficult to assay PDC kinase routinely in crude mitochondrial extracts using endogenous yeast PDC, purified exogenous yeast or bovine PDC as substrates with reproducible phosphorylation only occurring in intact mitochondria.…”

“…Attempts thus far to demonstrate PDC kinase activity in yeast have been unsuccessful [7]. However, the first indirect evidence that yeast PDC may be regulated in vivo by a phosphorylation-dephosphorylation mechanism was presented by Uhlinger et al [8].…”

Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system

“…The DHLTA of acetoin-grown cells of P. carbinolicus was purified 24-fold to electrophoretic homogeneity in a two-step procedure (Table 3). Because of the high sensitivity of DHLTA from P. carbinolicus to proteolytic digestion (see below), the purification of DHLTA was performed in the presence of the protease inhibitors EDTA, PMSF, leupeptin, and pepstatin (29).…”