The metabolism of the obligate anaerobe Clostridium kluyveri has for some years been the subject of detailed study by H. A. Barker and his colleagues at the University of California. In last year's Symposium on Phosphorus Metabolism, Barker (3) reviewed many aspects of this work. The present discussion will be limited to a brief summary of some results which are of interest in the general problem of fatty acid oxidation.

Stadtman and Barker (56,57) in an important series of papers have described the preparation of soluble, cell-free extracts from dried cells of C. kluyveri which are capable of oxidizing fatty acids in chain length up to 8 carbon atoms. These same extracts are also capable of carrying out the synthesis of fatty acids, and it appears that the process of oxidation is simply the reversal of the synthetic mechanism. Although the organism contains no cytochrome system, atmospheric oxygen can be used as electron acceptor, presumably because of the presence of large amounts of auto-oxidizable flavo-protein enzymes, and the reaction may be followed by measuring O2 consumption manometrically. Hydrogen peroxide does not accumulate when fatty acid oxidation is studied at pH 8.0 near the optimum hydrogen ion concentration for this reaction. Under these conditions, and in the presence of inorganic phosphate, butyrate is oxidized according to the following equation:

CH8CH2CH2COOH + P1 + O2-→ CH8COOH + CH8CO ~ P.

Acetate and acetyl phosphate accumulate as the products of this oxidation. Since these remarkable enzyme preparations also contain enzymes capable of activating molecular hydrogen, the above reaction may be reversed and fatty acid synthesis demonstrated simply by shaking acetyl phosphate and acetate in an atmosphere of hydrogen.

In these experiments of Stadtman and Barker, the crude bacterial extract carried out the oxidation of fatty acid without the addition of cofactors other than inorganic phosphate. The addition of magnesium ion, fumarate, or adenine nucleotide to the crude preparation did not increase the rate of oxidation. With dialyzed preparations, however, optimal oxidation could be observed only when the preparations were supplemented with acetyl phosphate and yeast extract. The cofactors present in the yeast extract were not further identified. In later experiments, Kennedy (24) studied the cofactor requirements for butyrate oxidation, using enzyme preparations from C. kluyveri which were resolved by treatment with acid ammonium sulfate. In these experiments, FAD, DPN, CoA, and divalent cation as well as acetyl phosphate were required to restore activity. Unfortunately, the ammonium sulfate treatment resulted in large losses of activity, and was thus wasteful of enzyme. It is likely that a full understanding of the role of the various cofactors in the bacterial enzyme system will be achieved only when the enzymes concerned are fractionated and single enzymatic transformations studied. Since the enzymes appear to be reasonably stable, and are devoid of complications such as the necessity for organized particulate structure, it appears likely that this goal will be ultimately achieved.

Since suspected intermediates of 4-carbon chain length are more readily available than for other fatty acids, special study has been devoted to oxidation and synthesis of butyrate in this system. Stadt-man and Barker (56) tested a number of possible intermediates. Of all compounds tested, only one, vinyl acetic acid (3-butenoic acid), showed the properties of a possible intermediate, namely, rapid reduction to butyrate when incubated anaerobically under hydrogen with the enzyme, and rapid oxidation to acetate and acetyl phosphate when tested aerobically in the presence of inorganic phosphate. Particular attention was paid to β-hydroxybutyrate and acetoacetate, and definite evidence was obtained which excluded these compounds as intermediates. Acetoacetate when added to the system at pH 8.0, optimal for the fatty acid synthesis, could be reduced only to β-hydroxybutyrate, which then showed no further reduction to butyrate. Crotonate and isocrotonate were completely inactive.

When isotope tracer experiments, designed to test the only active compound found, namely, vinyl acetate, were performed, the result obtained indicated surprisingly enough that this compound could not represent an obligate stage in butyrate synthesis or oxidation. When C14abeled acetate was anaerobically converted to butyrate in the presence of vinyl acetate, and the butyrate and residual vinyl acetate were isolated at the end of the experiment, the butyrate was found to contain considerable amounts of radioactivity, as was to be expected, but the vinyl acetate recovered was devoid of radioactivity. Clearly the butyrate could not have been formed from the acetate by a process including free vinylacetate as an obligate intermediate.

The results of Stadtman and Barker were confirmed in a later study by Kennedy and Barker (25), who also tested dl-a-hydroxy-butyrate, γ-hydroxybutyrate, γ-butyrolacetone, dl-threo-a,β-dihy-droxybutyrate, DL- β,γ-epoxybutyrate, succinic semialdehyde, succinate, and a-ketovalerate. The last compound was tested in place of a-ketobutyrate, which was not available. All of these compounds were inert.

Oxidation of Butyrate in the Absence of Phosphate. Stadtman and Barker (56) observed significant oxidation of butyrate only in the presence of added inorganic phosphate, under which conditions the butyrate oxidized could be quantitatively accounted for as acetyl phosphate and acetate. In the experiments of Kennedy and Barker (25), however, butyrate oxidation in the virtual absence of inorganic phosphate could be demonstrated. Under these conditions acetyl phosphate and acetate were no longer the principal end-products, but rather acetoacetate was shown to accumulate. This finding at first added to the confusion concerning possible intermediates in the reaction scheme, since acetoacetate was shown to result from the oxidation of butyrate in the absence of inorganic phosphate, while unequivocal evidence, discussed above, had ruled out the possibility that it was an intermediate in the pathway from butyrate to acetyl phosphate and acetate in the presence of inorganic phosphate.

The mechanism of acetoacetate formation in these experiments proved to be of interest. When carboxyl-labeled butyrate was oxidized to acetoacetate in the absence of inorganic phosphate, and the resulting acetoacetate analyzed, it was found that the radioactivity was contained only in the carboxyl carbon, the acetone moiety being completely inactive. These results are in contrast to studies with enzymes derived from animal tissues in which carboxyl-labeled fatty acid gives rise to acetoacetate in which the label is more or less randomized between the carboxyl and carbonyl carbon, as discussed in a previous section of this paper.

In attempting to formulate the over-all pattern of butyrate oxidation in C. kluyveri, Kennedy and Barker (25) found it necessary to account for three fundamental experimental observations..The first of these is the lack of possible free intermediates. Of the large number of compounds tested, only vinylacetic acid is reactive in this enzyme system, and isotope tracer studies demonstrate unequivocally that it cannot be a normal intermediate. Furthermore, it cannot undergo any simple transformation into the true intermediate, such as shift in the double bond or the addition of the elements of water, since the products of such reactions were tested and found to be inert.