The Pyruvate‐Dehydrogenase Complex from Azotobacter vinelandii

Abstract

The pyruvate dehydrogenase complex from Azotobacter vinelandii was isolated in a five‐step procedure. The minimum molecular weight of the pure complex is 600000, as based on an FAD content of 1.6 nmol · mg protein⁻¹. The molecular weight is 1.0–1.2 × 10⁶, indicating 1 mole of lipoamide dehydrogenase dimer per complex molecule. Sodium dodecylsulphate gel electrophoretical patterns show that apart from pyruvate dehydrogenase (M_r 89000) and lipoamide dehydrogenase (M_r monomer 56000) two active transacetylase isoenzymes are present with molecular weight on the gel 82000 and 59000 but probably actually lower. The pure complex has a specific activity of the pyruvate‐NAD⁺ reductase (overall) reaction of 10 units · mg protein⁻¹ at 25°C. The partial reactions have the following specific activities in units · mg protein⁻¹ at 25°C under standard conditions: pyruvate‐K₃Fe(CN)₆ reductase 0.14, transacetylase 3.6 and lipoamide dehydrogenase 2.9. The properties of this complex are compared with those from other sources. NADPH reduced the FAD of lipoamide dehydrogenase as well in the complex as in the free form. NADP⁺ cannot be used as electron acceptor. Under aerobic conditions pyruvate oxidase reaction, dependent on Mg²⁺ and thiamine pyrophosphate, converts pyruvate into CO₂ and acetate; V is 0.2 μmol O₂· min⁻¹· mg⁻¹, K_m (pyruvate) 0.3 mM. The kinetics of this reaction shows a linear 1/velocity – 1/[pyruvate] plot. K₃Fe(CN)₆ competes with the oxidase reaction. The oxidase activity ist stimulated by AMP and sulphate and is inhibited by acetyl‐CoA. The partially purified enzyme contains considerable phosphotransacetylase activity. The pure complex does not contain this activity. The physiological significance of this activity is discussed.