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Leucine dehydrogenase found in the vegetative cells and spores of various Bacillus species is capable of catalyzing a DPN3-dependent oxidation of the branched-chain amino acids leucine, valine, isoleucine and the straight-chain amino acids norvaline and norleucine. The data obtained support the contention that the oxidation of these substrates is due to the activity of a single enzyme, l-leucine dehydrogenase, and not to the nonspecific action of glutamic or alanine dehydrogenase. The enzyme has been purified 380-fold from Bacillus subtilis strain IRC 1 and is completely free of alanine and glutamic dehydrogenases. Some characteristics of the enzyme are reported.

Current observations on the inhibition of tumor growth by enzymes such as asparaginase [ 1,2], glutaminase [3] , arginase [4,5] and phenylalanine ammonia-lyase [6] , which catalyze the essentially irreversible degradation of amino acids, have stimulated the search for the antineoplastic activity of other microbial enzymes related to amino acid metabolism. The antitumor activities of a folate-cleaving bacterial enzyme, carboxypeptidase G, [7,8], and jack bean urease [9] , and the inhibition ofgrowth and DNA synthesis of tumor cells by ascorbic acid oxidase [lo] also have been recently reported. In the present communication we describe some studies on the antitumor activities of several bacterial enzymes, especially leucine dehydrogenase and isozyme A of glutaminase, which catalyze the reversible oxidative deamination of L-leucine and some other aliphatic amino acids in the presence of NAD [ 1 l] , and the deamidation of glutamine and asparagine [ 121, respectively.

Publisher Summary This chapter describes the assay, purification, and properties of L-leucine dehydrogenase. The reaction may be measured spectrophotometrically by utilizing the increase in absorption at 340 m μ due to the conversion of DPN + to diphosphopyridine nucleotide (DPNH) in the presence of L-leucine. One unit of enzyme is the amount causing an increase of 0.001 in extinction per minute in the oxidative deamination assay. Specific activity is expressed as units per milligram of protein. The enzyme reacts with L-leucine, L-valine, L-isoleucine, and L-norvaline. DPN is absolutely specific for the enzyme. The optimum pH of the oxidative deamination reaction is 11.3. The enzyme is very sensitive to sulfide and p-chloromercuribenzoic acid ( p CMB). The inhibition by the latter is completely reversed by L-cysteine. Chelating agents do not affect enzyme activity. The enzyme can also be assayed by the decrease in absorbance in the reductive amination at 340 m μ . This assay is more sensitive, but there is some interference by the DPNH oxidase and lactic dehydrogenase present in crude extracts.

The preparation of crystalline L-leucine dehydrogenase from the extract of Bacillus sphaericus is described. The enzyme is homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. The molecular weight is 280,000. The enzyme catalyzes the oxidative deamination of L-leucine, L-valine, L-isoleucine, L-norleucine and L-norvaline, while none of D-leucine, D-valine, L-alanine and L-glutamate is deaminated. NAD only serves as the hydrogen acceptor.

An enzyme present in Bacillus cereus has been found to catalyze the oxidative deamination of l -leucine. The reaction is freely reversible and similar to glutamic acid dehydrogenase. Diphosphopyridine nucleotide serves as the hydrogen acceptor. The pH optimum for oxidative deamination is 11.3. The equilibrium of the reaction favors synthesis of the amino acid by a reductive amination of its keto analog. The enzyme is inhibited by p -chloromercuribenzoate, and l -cysteine reverses this inhibition completely, suggesting that —SH groups are necessary for activity. A study of kinetics of the enzyme has been presented. The possible role of the enzyme in the metabolism of some species of Bacillus has been discussed.

In order to grow in a simple salts medium in which ammonia provides the sole source of nitrogen, micro-organisms must possess some mechanism for the synthesis of amino acids from ammonia and intermediary metabolites. In many bacteria this requirement is fulfilled by the enzyme glutamate dehydrogenase (EC 1.4.1.4), which reductively aminates 2-oxoglutarate to glutamate. In other bacteria (particularly in some species of Bacillus) analogous amino acid dehydrogenases (e.g. alanine dehydrogenase and leucine dehydrogenase) functionally replace glutamate dehydrogenase and glutamate is then formed by an aminotransferase reaction. Available kinetic data indicate that amino acid dehydrogenases generally have high Km values for ammonia (above 5mM; see Sanwal & Zink, 1961; Wiame, Pierard & Ramos, 1962), and one might therefore expect that when bacteria are growing in a simple salts medium the intracellular free ammonia concentration always would be relatively large. When growth takes place in the presence of a large excess of ammonia, the intracellular free ammonia content is difficult to assess without washing the organisms (which is undesirable, since it may cause changes in 'pool' composition; see Britten, 1965). But, when organisms are cultured in a chemostat under conditions where growth is limited by the availability of ammonia, the intracellular free ammonia can be easily determined by extracting the organisms with 0.25% HC104 and applying the extracts to the columns of a Technicon automatic amino acid analyser; washing these organisms is unnecessary since the extracellular free ammonia concentration is negligible (Herbert, 1958). With ammonia-limited chemostat cultures of Aerobacter aerogenes, growing at a dilution rate of 0.3h-1 in the medium of Tempest (1965) at pH6.5 and 35°C, the intracellular free ammonia concentration was found to be less than 0.005% of the bacterial dry weight * Present address: Novo Industri A/S, Fuglebakkevej 115, 2200 Copenhagen N, Denmark. (below 0.5mM), well below theKm value for amnmonia (3-4mM) of A. aerogenes glutamate dehydrogenase. This observation prompted a detailed quantitative study of the relationship between growth condition and bacterial glutamate dehydrogenase activity of