THE CYCLOPHORASE COMPLEX OF ENZYMES

SUMMARY 1 The cyclophorase complex of enzymes which implement the citric acid cycle is contained within the mitochondrial bodies. 2 Mitochondria behave as fibre-like macromolecules which can be comminuted first to submicroscopic particles (microsomes) and then eventually to soluble proteins. 3 The cyclophorase system of enzymes can be prepared by differential centrifugation of mitochondria at low temperatures. Even at oo the cyclophorase activity declines sharply in a matter of hours. 4 The freshly prepared enzyme gel contains all the necessary co-factors and enzymes for the reactions of the citric acid cycle and of fatty acid oxidation. With time, a requirement for adenosine-5-phosphate and magnesium ions emerges. 5 Various amino-acids and fatty acids (even-numbered) are burned to carbon dioxide and water in the cyclophorase system by virtue of their ability to give rise to members of the citric acid cycle, either directly or indirectly. 6 Although intermediates do not accumulate during the normal activity of the cyclophorase system, experimental devices can be employed which permit the study of one-step oxidations or conversions. 7 Fatty acids are not oxidized unless ‘sparked’ by the co-oxidation of some member of the citric acid cycle. The sparking effect is eliminated by either 2:4-dinitrophenol or gramicidin. 8 The oxidation of fatty acids can be shown to proceed by way of β-oxidation. The β-keto acids interact with oxalacetate in a transacetylation reaction leading to formation of citric acid and a fatty acid with two carbon atoms less than the parent acid. 9 Under conditions for active oxidation acetoacetic acid arises in liver cyclophorase principally if not exclusively as the terminal 4-carbon residue of the even-numbered fatty acids. Odd-numbered fatty acids from C3 to C7 do not give rise to acetoacetic acid in significant amount, but C9 and C11 acids do. Propionic acid which arises as the terminal residue of the odd-numbered fatty acids is inert in kidney, but is oxidizable in liver to carbon dioxide and water by way of pyruvic acid as intermediate. In presence of malonate or under conditions where the citric acid cycle is inhibited, both odd- and even-numbered fatty acids can be converted quantitatively to acetoacetic acid in liver cyclophorase. 10 The cyclophorase system catalyses a group of synthetic condensations which are all distinguished by (1) the necessity for sparking by some members of the citric acid cycle, and (2) inhibition by 2:4-dinitrophenol and gramicidin. 11 The oxidases which implement the oxido-reductions of glycolysis are present at least in part in the cyclophorase complex of rabbit kidney and liver. None of the other enzymes of the glycolytic system occur in association with mitochondria. 12 The pyridinoprotein enzymes of the cyclophorase complex appear to be firmly linked with their prosthetic groups. With the transition from particulate to soluble pyridinoproteins, there is a loss of the capacity to bind the prosthetic group. The classical, soluble pyridinoprotein enzymes, with one exception, are fully dissociated with respect to the pyridinenucleotide prosthetic group. 13 The cyclophorase gel contains substantial amounts of the pyridinenucleotides, flavindinucleotides, thiaminephyrophosphate, the acetylation coenzyme and the adenosine polyphosphates, but a relatively lower concentration of pyridoxal phosphate and folic acid. 14 The principal elements in the electron transfer sequence between substrate and molecular oxygen in the cyclophorase complex appear to be (a) the pyridinoproteins, (b) the cytochrome reductases and (c) the cytochrome oxidase system of Keilin. 15 During active oxidation of some members of the citric acid cycle, inorganic phosphate becomes esterified and eventually accumulates as inorganic pyrophosphate. When oxidation is carried out in presence of the glucose-hexokinase system, the esterification of inorganic phosphate leads to the accumulation of glucose-6-phosphate. 16 Inorganic phosphate can be shown to be essential for maximal activity of most of the oxidases of the cyclophorase system. Experimentally, it has proved difficult to reduce the level of inorganic phosphate in the enzyme gel to the point of zero oxidative activity in absence of added inorganic phosphate. This residual activity can be referred in part to a reservoir of potential inorganic phosphate in the gel. There remains, however, the possibility that inorganic phosphate is necessary, not in the primary oxidative step, but in subsequent steps which involve regeneration of the coenzyme. 17 During the oxidation of malate, citrate, pyruvate, glutamate, proline, β-hydroxybutyrate and α-ketoglutarate, probably three molecules of inorganic phosphate become esterified per atom of oxygen absorbed; whereas the P/O ratio for the oxidation of succinate is probably 2. The efficiency of oxidative phosphorylation is about 60%. 18 Radioactive inorganic phosphate is rapidly taken up by the gel during active oxidation in a form which resists leaching out by exhaustive washing. Evidence is presented in favour of the hypothesis that the compound responsible for the incorporated radioactivity is of the nature of a labile phosphoric ester (gel P). In all of the available analytical procedures applied for estimation of labile phosphoric ester, gel P is estimated as inorganic phosphate. It is distinguishable from inorganic phosphate only on the basis of properties exhibited in the gel. 19 In addition to the labile gel P, there is evidence of one or more stable pyrophosphoric esters in the gel which become radioactive when the gel is allowed to carry on oxidation in presence of radioactive inorganic phosphate. Adenosine di-and triphosphates have been shown to be constituents of the pyrophosphate fraction. 20 In presence of 2:4-dinitrophenol, the requirement for inorganic phosphate appears no longer to obtain. This observation has led Lomis & Lipmann to postulate the uncoupling of oxidation and phosphorylation. Evidence is presented that dinitrophenol merely uncouples oxidation and transphosphorylation. By appropriate devices the requirement for inorganic phosphate even in presence of dinitrophenol is demonstrable. 21 Evidence is accumulating that the pyridinenucleotides and flavindinucleotide are involved in the process by which inorganic phosphate becomes esterified in the cyclophorase system. The formation of stable phosphoric esters of the various substrates which give rise to oxidative phosphorylation appears to be excluded. 22 Oxidative phosphorylation with a P/O ratio greater than 1 is a phenomenon characteristic only of the intact mitochondrial unit. The microsomes which arise from mitochondria (prepared from kidney or liver) by a process of comminution have lost completely the capacity for esterifying inorganic phosphate. 23 The cyclophorase system contains the full enzymatic apparatus for carrying out certain synthetic condensations. Reactive forms of acetyl, benzoyl, phenol, carbon dioxide, glutamic acid and β-keto acids must be generated in order to have these condensations take place. The acetylation coenzyme of Lipmann appears to be intimately involved in all reactions involving activated molecules. 24 Some consideration is given to the type of process which may underlie the formation of activated molecules. The cyclophorase system appears to make use of chemical principles which are not applicable to the enzymes derivable from the complex by isolation procedures. 25 There is a wide spectrum in the ease with which various enzymes separate out from mitochondria. This behaviour makes it difficult to draw a sharp line of demarcation between the enzymes which properly belong to the mitochondrial complex, and those which are either adsorbed or loosely held. 26 Some examples are presented of the intimate relation between mitochondria and physiological function in muscle and kidney.