lyzes a slow deamination of adenine and guanine. 118 The products of hypo
(51) RCOCOOH + COOHCH 2 CH 2 CHNH 2 COOH
COOHCH2CH2CHNH2COOH + H20 + DPN+ ^±
(52) COOHCH2CH2COCOOH + N H3 + D P N H + H+
T A B L E X I V
AMINO ACIDS USED AS OXIDANTS IN THE STICKLAND REACTION BY Clostridium sporogenes
Compound Relative ratea
Glycine 100
Proline 100
Hydroxyproline 100
Ornithine 100
Arginine 80
Tryptophan 67
Tyrosine 25
Cysteine 22
Cystine 16
Methionine 15
Aspartate 2
β The figures give the relative rates (glycine = 100) of hydrogen uptake, measured manometrically, in the presence of a washed suspension of C. sporogenes and the in
dicated amino acids.1 3
of phosphate results in formation of acetyl phosphate, presumably b y t h e action of t h e enzyme phosphotransacetylase. Therefore, both high energy acetyl and phosphoryl groups are available for synthetic reactions. T h e oxidation of higher α-keto acids in t h e presence of phosphate also results in formation of t h e corresponding acyl phosphates.
B . REDUCTIONS
T h e most distinctive p a r t of t h e Stickland reaction is t h e apparently direct reduction of certain amino acids, including glycine, proline, hydroxy-proline, a n d ornithine (Table X I V ) . This type of reaction has been observed only in certain Clostridia.
T h e reductions of glycine to acetic acid a n d ammonia and of proline t o δ-aminovalerate during t h e coupled reaction with alanine have already been mentioned. Cells of C. sporogenes, grown in t h e presence of glucose, catalyze t h e same reductions using gaseous hydrogen as reductant.1 3 T h e r a t e of hydrogen u p t a k e can be followed manometrically. This provides a convenient a n d moderately sensitive method for identifying compounds t h a t can be used as oxidants b y C. sporogenes.
B y this method t h e amino acids listed in Table X I V , and, in addition, a considerable number of non-nitrogenous compounds were shown t o serve as oxidants. T h e reduction of hydroxyproline was shown t o require one mole of hydrogen a n d t o occur without t h e formation of ammonia; t h e presumed reduction product, 7-hydroxy-5-aminovaleric acid, was not
di-rectly identified. Ornithine is reduced to δ-amino-n-valeric a c i d1 3'3 6 accord
ing to t h e equation:
N H2( C H2)3C H N H2C O O H ·+ H2 -> NH2(CH2)4COOH + N H3 (53) Ornithine δ-Aminovaleric acid
T r y p t o p h a n is apparently reduced by hydrogen t o indolepropionic acid - C C H2C H2C O O H
C C H2C H N H2C O O H
II
CH + H2 i y J v^ / C H + N H , (54) N H N H
Tryptophan Indolepropionic acid
and ammonia.1 2 A derivative of indolepropionic acid was isolated and par
tially characterized. T h e expected stoichiometry according to equation (54) was not observed because t r y p t o p h a n was simultaneously fermented to other products.
T h e enzymic reactions in proline reduction by C. sticklandii have been partially analyzed. Crude cell-free extracts catalyze a reduction of DL-proline by several dithiol compounds1 6 1 according to equation (55). T h e most effective dithiol so far tested is 1,3-dimercaptopropanol. Several other
DL-proline + R ( S H )2 —> δ-aminovalerate + R S2 (55) dithiols, including 6,8-dimercapto-octanoate (reduced lipoic acid) and
1,2-dimercaptopropanol (BAL), are somewhat less active whereas monothiols like 2-mercaptoethanol are virtually inactive. Probably none of these com
pounds is the physiological reducing agent, since to be effective even 1,3-dimercaptopropanol must be used in a high concentration t h a t is unlikely t o occur in living cells. Furthermore, t h e reduction of proline by intact bacteria is relatively insensitive to inhibition by arsenite, whereas m a n y reactions involving dithiols are extremely sensitive t o this inhibitor. T h e physiological reducing agent has not been identified, b u t it is not reduced diphosphopyridine nucleotide.
T h e reduction of L-proline in t h e cell-free system apparently involves its conversion t o D-proline [reaction (56)] followed by t h e reduction of t h e
L-proline D-proline (56) D-proline 4- R ( S H )2 —• δ-aminovalerate -f R S2 (57)
latter t o δ-aminovalerate [reaction (57)].1 6 2 T h e enzyme systems catalyzing these two reactions have been separated. Reaction (56) probably involves more t h a n one enzyme, since there is evidence for t h e accumulation of an intermediate t h a t has some b u t not all of t h e properties of D-proline.1 6 3 T h e partially purified enzyme catalyzing reaction (57) cannot reduce L-proline.
Mg4"4", Ca4"4", and M n4 4 stimulate t h e reduction of D-proline. There is no clear evidence for participation of either D P N or pyridoxal phosphate in t h e reaction.
T h e reduction of glycine b y extracts of C. sticklandii can also be coupled with t h e oxidation of dithiols, b u t t h e reaction is more complex t h a n t h e reduction of D-proline. A soluble enzyme system responsible for t h e reduc-tion of glycine by 1,3-dimercaptopropanol appears to require orthophos-p h a t e and a orthophos-phosorthophos-phate acceorthophos-ptor such as adenosine monoorthophos-phosorthophos-phate ( A M P ) or A D P for full a c t i v i t y .1 6 3'1 6 4 T h e orthophosphate and phosphate acceptor can be replaced b y arsenate as in t h e triose phosphate dehydrogenase reac-tion. When orthophosphate (Pi) and A D P are used, one mole of A T P is formed per mole of glycine reduced in accordance with reaction (58). T h e
CH2NH2COOH + R ( S H )2 + Pi + ADP -» CH3COOH + N H8 + R S2 + ATP (58)
fact t h a t arsenate can substitute for phosphate and A D P indicates t h a t a high energy phosphoryl compound probably is formed as an intermediate and normally transfers its phosphoryl group t o A D P . T h e identity of this intermediate has not been established b u t evidence for t h e existence of an intermediate between glycine and acetate has been presented.
T h e enzyme preparation responsible for t h e reduction of glycine also reduces proline. I n t h e latter process, phosphate is not required and, when present, is not esterified. T h e difference between glycine and proline reduc-tion, with respect t o phosphorylareduc-tion, m a y or m a y not also exist in intact bacteria. At present there is no indication t h a t glycine is more effective t h a n proline as an oxidant for growing cells.
IV. Conclusions
T h e energy metabolism of anaerobic organisms involves a coupling of t h e oxidation and reduction of organic substrates or products of substrate transformation. T h e oxidation reactions are frequently similar or identical to corresponding reactions catalyzed b y aerobic organisms except for cer-tain limitations imposed b y t h e absence of oxygen. For example, t h e oxida-tive deamination of amino acids and t h e oxidaoxida-tive decarboxylation of keto acids occur in much t h e same ways in b o t h aerobic and anaerobic species.
However, t h e oxidation of aromatic compounds, like tyrosine, b y anaerobic species appears t o be generally restricted t o an a t t a c k on t h e aliphatic side chain probably because of t h e need for direct participation of oxygen in hydroxylation and rupture of t h e benzene r i n g .1 6 6 Aliphatic compounds are commonly oxidized t o fatty acids which accumulate in t h e medium. F a t t y acids are generally not decomposed b y fermentative bacteria apparently because t h e first step in their oxidation occurs a t such a high oxidation-reduction potential t h a t it cannot be effectively coupled with other available
oxidants. B u t aside from such limitations there is nothing distinctive about oxidations catalyzed b y anaerobic bacteria.
T h e reduction reactions of fermentative organisms are more distinctive.
T h e problem of providing organic oxidants has been solved in a variety of ways. Some species use a substrate directly as a n oxidant. This is done, for example, b y Clostridium sporogenes and other organisms t h a t are capable of reducing glycine t o acetic acid, proline t o δ-aminovaleric acid, or t r y p t o p h a n t o indolepropionic acid. Similarly, t h e purine-fermenting Clostridia reduce uric acid t o xanthine, and Zymobacterium oroticum reduces orotic acid t o dihydro-orotic acid. Other species transform t h e nitrogenous sub
strate t o a non-nitrogenous intermediate t h a t is capable of serving as an oxidant. Examples of this are t h e conversion of serine t o propionate, prob
ably via pyruvate, b y C. propionicum, and t h e conversion of glutamate t o b u t y r a t e via pyruvate and presumably acetyl-CoA b y C. tetanomorphum.
Another common method of accomplishing a n anaerobic oxidation is b y the removal of gaseous hydrogen. A striking example is t h e fermentation of threonine b y Micrococcus aerogenes in which t h e oxidation of one mole of substrate t o propionate and carbon dioxide is coupled with t h e forma
tion of one mole of hydrogen. This method of removing electrons is ob
viously only applicable when t h e reducing system operates a t a n oxidation-reduction potential well below t h a t of t h e hydrogen electrode.
T h e fermentation reactions discussed in this chapter differ markedly with respect t o t h e number of reactions t h a t occur before t h e nitrogen is removed from t h e substrate. I n several fermentations t h e nitrogen is re
moved in t h e first step. Examples of this are t h e nonoxidative deaminations of serine, threonine, and cysteine, t h e reductive deaminations of glycine and tryptophan, and t h e oxidative deamination or possibly transamination of various amino acids b y bacteria utilizing t h e Stickland reaction. I n other instances, one or more of t h e nitrogen atoms of t h e substrate are retained through a sequence of two or more reactions. Examples are t h e fermenta
tions of purines, pyrimidines, allantoin, arginine, histidine, and glutamate.
I n most fermentations of nitrogenous compounds much or all of t h e nitro
gen is ultimately converted t o ammonia or urea. However, in a few fermen
tations some of t h e final products still retain a nitrogen atom. Some exam
ples are t h e accumulation of oxamic acid in t h e fermentation of allantoin b y Streptococcus allantoicus, of glycine in t h e fermentation of purines b y C. cylindrosporum, of indolepropionic acid in t h e fermentation of t r y p t o p h a n by C. sporogenes, and of δ-aminovalerate in t h e reduction of proline or or
nithine b y t h e same organism.
Only a few of these fermentations have been studied t o t h e extent t h a t we have a fairly comprehensive knowledge of all of their chemical steps.
T h e Stickland reaction, and t h e fermentations of purines and glutamate
are perhaps best understood, b u t conspicuous gaps exist in our knowledge of even these processes. I n t h e Stickland reaction t h e mechanism of the reduction of glycine to acetic acid, coupled with the esterification of phos
phate, is still obscure. In t h e purine fermentation, t h e p a t h of acetate synthesis from carbon dioxide and t h e mechanism of the coupling between glycine or pyruvate oxidation and uric acid reduction are not understood.
I n t h e glutamate fermentation b y C. tetanomorphum t h e novel reactions responsible for the conversion of glutamate t o 0-methylaspartate and the specific role of t h e pseudovitamin Βχ2 coenzyme remain t o be elucidated.
Knowledge of t h e chemistry of most of t h e other fermentations is still highly fragmentary, in several instances consisting only of t h e quantitative identification of t h e products. Some fermentations of nitrogenous com
pounds undoubtedly still await discovery. Obviously this is an area which has not been thoroughly explored and in which significant discoveries can still be made.
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