Johannes Ullrich

Subunit size of cytoplasmic yeast pyruvate decarboxylase

From the number of thiamine pyrophosphate (TPP) binding sites estimated earlier [l] , cytoplasmic yeast pyruvate decarboxylase (PDC) (EC 4.1.1.1) was hitherto expected to contain 4 polypeptide chains of a molecular weight of 40,000-45,000, each of them carrying one active site. For the decarboxylase moiety in the pyruvate dehydrogenase complex of E. coli, Reed et al. found a similar molecular weight of the intact enzyme [2] and an a,cw,/3;&structure [3]. In contrast, cytoplasmic yeast pyruvate decarboxylase was found to be separable into only two polypeptide chains with a molecular weight of circa 90,000 each, which are probably identical.

Activity stain for pyruvate decarboxylase in polyacrylamide gels

A method for the localization of pyruvate decarboxylase bands in polyacrylamide gels after electrophoresis using 1,2-dianilinoethane in dilute acetic acid as reagent for acetaldehyde formed from pyruvate by the holoenzyme is described. A white condensation product forms in the bands and precipitates within a few minutes. The more or less opaque bands can be viewed and photographed against a dark background and scanned in a densitometer. The detection limit is at about 10 mU pyruvate decarboxylase when the gels are bathed in the staining solution for 20 min. Several other methods were tested and failed to produce satisfactory results.

Amino acid composition of α- and β-chains of yeast and wheat germ pyruvate decarboxylase

Thus, the recently isolated structural gene for yeast PDC cannot suffice for coding both chains.

Thiamine Pyrophosphate-Catalyzed Enzymatic Decarboxylation of α-Oxo Acids

Publisher Summary This chapter focuses on thiamine pyrophosphate-catalyzed enzymatic decarboxylation of α-oxo acids. The first enzymatic decarboxylation of an α-oxo acid was described as: a fermenting extract from brewers yeast cleaves pyruvate into acetaldehyde and carbon dioxide. Later, it was found that besides the protein of the enzyme, a thermostable cofactor (“cocarboxylase”) is necessary for the enzymatic cleavage of pyruvate. For decarboxylation of other α-oxo acids to the corresponding aldehydes, for their oxidative decarboxylation leading to acyl coenzyme A derivatives, and for a number of other enzymatic reactions, the same coenzyme was found to be necessary. By comparison of thiamine catalysis with that of cyanide in the acyloin condensation, Breslow came to the conclusions that thiamine forms a 2-carbanion with a p K comparable to that of HCN; the upper limit found for the p K was 20. The exclusively kinetic measurements could not account for activation energy, and therefore the p K of thiamine is expected to be several units lower. In this chapter, mechanism of nonenzymatic thiamine (Thiazolium) catalysis are analyzed in detail. Isolation of 2-α-hydroxyalkyl derivatives of thiamine pyrophosphate from enzymatic incubation mixtures is described. Effects of substituents at the thiazolium ring are also discussed.

Amino acid composition of cytoplasmic yeast pyruvate decarboxylase

Fyruvate decarboxytase was isolated essentially as described earlier [l] from Saccizaronz,wes carlsbergensis: (a) fresh brewer’s yeast, strain Weihenstephan T34, obtained from Canter-Brauerei, D-78 Freiburg, with a slight modification of the get filtration procedure: (b) dried brewer’s yeast, regular strain, obtained from Anheuser-Busch Inc., St. Louis. MO., USA. The best fractions of 3 different enzyme preparations from source (a) and one from source (b) were exhaustively diatysed against water at 3” for 2---3 days and freeze-dried. Two thirds of the original specific activity of 62, 59, and 74 units/mg [t] for (a) (the highest specific activity reached as yet was 85 units/ mg) and 60 units/mg for (b) were lost during this procedure, the apoenzyme lost all activity by this treatment. 7-10 mg of the resulting hygroscopic white

[45] Enzymatic preparation, isolation, and identification of 2-α-hydroxyalkylthiamine pyrophosphates

Publisher Summary This chapter discusses the enzymatic preparation, isolation, and identification of 2-α-hydroxyalkylthiamine pyrophosphates. 2-Hydroxymethyl-thiamine pyrophosphates (TPP), 2-α-hydroxyethyl-TPP, 2-α,β-dihydroxyethyl-TPP, and 2-α-hydroxy-γ-carboxypropyl-TPP have been isolated from the incubation mixtures of TPP-dependent enzymes. These compounds are discussed in the chapter to represent the TPP-activated aldehyde intermediates of the enzymatic reactions. In the case of 2-α-hydroxyethyl-TPP, however, there is substantial evidence that the nucleophilic α-carbanion that arises from the decarboxylation step in the yeast pyruvate decarboxylase reaction is protected from protonization by the lipophilic environment of the active site. Thus, the α-carbanion of 2-α-hydroxyethyl-TPP is considered the real activated intermediate. The crude preparations of mitochondrial α-oxoacid dehydrogenase complexes are prepared almost free of nicotinamide adenine dinucleotide (NAD + ) and coenzyme A (CoA). Such preparations still decarboxylate the α-oxoacids but are unable to oxidize the TPP-linked decarboxylation products. The α-carbanions of the 2-α-hydroxyalkylthiamine pyrophosphates add a proton and are replaced at the active site of the enzyme by free TPP.