VITAMINS as COENZYMES HENRY TAUBER * The McLeod Infirmary. Florence, South Carolina INTRODUCTION
M
of the carboxylase system manganese salts are most effective.
ANY years before anything was known about the chemistry of vitamins the interest of biological chemists centered around typical biocatalysts such as cozymase which was found to be identical with diphosphopyridine nucleotide, cocarboxylase with vitamin B, pyrophosphoric acid ester, hexuronic acid with vitamin C, and cytoflav with lactoflavin monophosphoric acid ester. Some of these substances were first known as coenzymes. Coenzymes may be defined as heat-stable crystalloidal organic compounds which are specific and indispensable components of one or more enzyme systems. Only minute amounts of a coenzyme are required to complete an enzyme system. Enzymes are catalysts which are produced by the living cell, but whose action is independent of the living cell, and which are destroyed if their solutions are heated. Many appear to be of protein nature. Ten enzymes have been isolated in recent years in crystalline form. They are all proteins (1, 2 ) . Some enzymes require minute amounts of activators for their activity whereas others requirePoth activators and coenzymes. Activators are also heat-stable crystalloids. They may be replaced and, therefore, are nonspecific; e. g., hydrogen chloride, sodium chloride, and hydrocyanic acid. Hydrogen chloride, the activator of pepsinogen, converting the inactive precursor into the active enzyme pepsin, may be replaced by any acid, inorganic or organic. The hydrogen ions are the activating factors (2). Sodium chloride, the activator of pancreatic amylase (a starch-splitting enzyme), may be replaced by any of a large number of salts. The dominating factors here, however, are the anions. Chlorides activate best. whereas sulfates are inert. In the case
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*Present address: ResearchLaboratory, Johnsonand Johnson, New Brunswick, New Jersey.
* I t is the author's aim to point out the interaction of certain enzymes with certain vitamins. It should be noted, however, that this paper is not devoted to human metabolism only.
In 1911 Neuberg (3) discovered that yeasts and plants contain an enzyme which decarboxylates a-keto acids such as pyruvic acid, converting them into the corresponding lower aldehyde; e. g., acetaldehyde. In mammals and certain bacteria, however, pyruvic acid is oxidized differently than in plants. Decarboxylation occurs with simultaneous dehydrogenation (4). The enzyme concerned in this reaction is called pyruvic acid dehydrogenase. Only in 1932 was it proved by Auhagen (5) that carboxylase requires a coenzyme. He has shown when brewers' yeast was washed with an alkaline phosphate solution i t lost its power to attack pyruvic acid and that after the addition of the coenzyme, which he obtained in a semipure state from yeast, the carboxylase became active again. For full activity, however, the addition of a trace of magnesium sulfate was required. This was known to be also the case with zymase. Auhagen did not study the chemical composition of his cocarboxylase in great detail. He knew, however, that i t was not identical with any of the known constituents of yeast and suggested that the substance was probably a phosphoric acid ester of an organic compound. Simola (6) in a paper entitled "On the Cozymase and Cocarboxylase Content of the Rat Organism in B-avitaminosis" reported very important findings which pointed to a connection between vitamin Bi and cocarboxylase. He obtained a sample of Auhagen's cocarboxylase and found that i t possessed vitamin B, potency on rats. Tissues of rats kept on a vitamin B,
up to ten grams, and, as would be expected, many grams of living yeast (11) would pbosphorylate faster than a few hundred mg. of dry yeast. ISOLATION AND IDENTIFICATION OF COCARBOXYLASE I have shown that a phosphatese of the duodenal Recently Lohmann and Schnster (7) have isolated co- mucosa of the pig has the ability to phosphorylate carboxylase in pure crystalline state and found that i t thiamine. Cocarboxylase formation is considerable is the pyrophosphoric acid ester of thiamine (vitamin even in the absence of added inorganic phosphate, indicating that antolyzing cells furnish a phosphate Bd : donator for this reaction (8). After twenty-four hours C? ?H ?H of incubation the cocarboxylase is closely bound to N=C-NHX ~ = = = c - c H ~ c H ~ -the+insoluble L I ~enzyme - o Hprotein material and is only reII II I I / leased after four minutes of boiling (12). The super0 0 H 8 C 4 C-CH2-N natant fluids of unboiled samples are inactive. While II II / 1 N-CH C1- C H - 4 the duodenal enzyme vrevaration has the ability to The isolation of cocarboxylase from yeast is very convert thiamineto co>&boxylase under a variety of laborious and difficult. The yield is poor. Only fifteen conditions, the enzyme preparation cannot hydrolyze per cent. of the cocarboxylase present in the first extract the coenzyme. This indicates that the phosphorylation may be recovered. Lohmann and Schuster obtained is catalyzed by an enzyme system, phosphatese different 700 to 800 milligrams per 100 kilos of yeast. A few from the phosphatases which hydrolyze esters of phosphoric acids. Cocarboxylase, however, is rapidly hymicrograms, however, suffice for an enzymic test. drolyzed by kidney tissue. PROPERTIES OF COCARBOXYLASE Lipschitz and associates (13) constructed a system Cocarboxylase is a very stable Hydrolywhich thiamine be very phosphosis with N hydrogen chloride for fifteen minutes liberates rylated yeast. Their fivemicroand the coen- grams of thiamine, boiled liver extract, hexosediphosone of the twophosphoric acid zyme becomes inactive. The second phosphoric acid phate, magnesium, manganese, phosphate buffer, and acid. Without pyruvic acid cOcarbOxylase group, however, is removed only after five hours of CPwithin sixty minutes, is very slight or comboiling with N~~~~~~~chloride, Hydrolysis with N / ~formation, ~ pletely lacking. hydrogen chloride for sixty minutes inactivates only fifty-five per cent. of the cocarboxylase and a correspondINHIBITORS OF ENZYMIC COCARBOXYLASE SYNTHESIS ing amount of phosphorus is removed (7). Experiments similar to those which lead to the I have found (14) that cell poisons such as toluene chemical identification of thiamine* had been con- and chloroform, and so forth, completely inhibit the ducted by Lohmann and Schuster with cocarboxylase. action of the enzyme phosphatese. Small amounts of They identified the nucleus of cocarboxylase with toluene, however, do not effect the enzyme carboxylase. thiamin, and found that in the pigeons' test cocarbox- According to Lipschitz and co-workers (15) 0.005 M ylase is almost twice as active as thiamin. iodoacetate inhibits completely cocarboxylase formaCocarboxylase is also the coenzyme of pyruvic acid tion from thiamine by atiozymase. Carboxylase, howdehydrogenase, and because thiamine cannot be syn- ever, is not affected, so that performed cocarboxylase thesized and stored by the body a constant supply is may readily be determined by this method. necessary. In thiamine deficiency pyruvic acid increases ACTIVATORS OF THE CARBOXnASE-COCARBOXYLASE in the blood, which diminishes very rapidly when the SYSTEM vitamin is supplied. At the same time certain pathological systems also disappear. Auhagen (5) has shown that similar to cozymase activity the carboxylase-cocarboxylasesystem also reENZYMIC SYNTHESIS OF COCARBOXYLASE FROM quires magnesium ions for full activity. Lohmann and THIAMINE Schuster (7) found that manganese ions are much better Shortly after the publication of the paper by Loh- activators than magnesium ions. Much smaller mann and Schuster (7) simultaneous and independent amounts of manganese salts are required to obtain full reports appeared from three different laboratories on activity. In addition to magnesium and manganese the enzymic synthesis of cocarboxylase from thiamine. salts I have tested a series of other salts and found that Washed cocarboxylase-free dry brewers' yeast, and sodinm sulfate, sodium chloride, potassium chloride, duodenal phosphatese (8); dry brewers' yeast with and sodium cyanide also activate this enzyme system hexosediphosphate, and adenosintriphosphate (9) ; and whereas sodium nitrate does not (16). Magnesium and living yeast (10) were employed, respectively. The manganese salts activate best. While sodinm cyanide yeast preparations used for the phosphorylation of a activates some enzymes, i t inhibits others. Sodium few mg. of thiamine varied from a few hundred mg. cyanide appears to be a good activator of the carbox'The chemistrp of thiamine has been recently reviewed by ylase system. It activates probably because i t conv a t s pyruvic acid into a cyanohydrin (enol). WILLWS. R . R.,Erg. Vit. Harmon Forrchung, 1, 213, 1938. free diet were poorer in cocarboxylase than those of an animal which received cocarboxylase.
1
SYNTHESIS OF COCARBOXYLASE FROM THIAMINE
Stern and Hofer (17) attempted to convert thiamine into its pyrophosphoric acid ester by treatment with phosphorus oxychloride. While this reagent was very useful in the conversion of lactoflavin (vitamin Ba) into flavin monophosphoric ester (IS), which is one of the coenzymes of the yellow oxidation enzyme of Warbnrg and Christian, only a very small amount of the thiamine could be phosphorylated by phosphoryl chloride. Apparently phosphorus oxychloride is not an efficient reagent for the introduction of the pyrophosphate group. Nevertheless, Stern and Hofer have shown by cataphoretic tests that conversion of the vitamin to its diphosphoric ester took place. I have been able to convert thiamine completely into the pyrophosphoric acid ester by phosphorylation with a mixture of ortho- and pyrophosphoric acid (19). The ester was isolated in pure, crystalline state (20). Hydrolysis and cleavage products have shown that the synthetic product is in every respect identical with the natural coenzyme. Cocarboxylase may be completely oxidized to its thiochrome compound by potassium ferricyanide and NaOH. The ferrocyanide produced during the reaction may be converted to Prussian blue and measured colorimetrically (21). This method may only be used, however, in the absence of reducing substances. The thiochrome compound also forms when excessive sodium hydroxide alone is added. The reaction is reversed on acidification and the cocarboxylase loses none of its activity. I found (19) that cocarboxylase (10 micrograms) gives a yellow color with the formaldehyde-azo-test of Kinnersley and Peters (22). Thiamine gives a red color.
coenzyme. If the cocarboxylase concentration is increased three times, carbon dioxide formation is only doubled, after the first fifteen minutes of the experiment. Recently Ochoa (22a) found that the action of pure (natural) cocarboxylase is accelerated by synthetic thiamine and by pyrimidines. This observation explains why impure synthetic cocarboxylase preparations were found to be just as active as pure ones (16a, 19). THE YELLOW OR FLAVIN ENZYME SYSTEM
In 1932 Szent-Gyorgyi and co-workers obtained from heart muscle a yellow water-soluble dye which they called cytoflav. The dye could readily be reduced to the leuco form and reoxidized to its original state by molecular oxygen. No evidence, however, was furnished by the Hungarian workers as to the physiological function of the new compound. Shortly after the discovery of cytoflav, Warburg and Christian announced isolation of a "yellow enzyme" from bottom yeast. The aqueous solutions of this enzyme are yellow and become colorless on reduction. The yellow color returns if the solution is shaken with oxygen. The two dyes are very closely related. Cytoflav is lactoflavin monophosphoric acid
I I
HOGH 6.7-dimethyl-9-d-ribitylisoalloxazin5'-phosphoric arid
CHe I
SPECIFIC FUNCTION OF COCARBOXYL.ASE
It is not known why cocarboxylase is necessary for carboxylase activity. I have shown that cocarboxylase has a protective action on carboxylase (16, 16a). The enzyme carboxylase when freed of its coenzyme becomes very labile. Cocarboxylase prevents it from rapid destruction. ESTIMATION OF COCARBOXYLASE ACTIVITY
The ingenious Warburg-Barcroft apparatus may be employed in all of the studies described in this paper. For carboxylase studies the method of Lohmann and Schuster (7) is very convenient. It should be noted that the carboxylase, as well as the cocarboxylase, content of the various yeasts d i m . The substrate concentration employed influences the reaction very greatly. The carbon dioxide formation increases when the substrate concentration is increased. For instance 2.5 mg. pyruvic acid will form 46 cmm. of carbon dioxide, 5 mg. 75 cmm., and 50 mg. 153 cmrn., in sixty minutes under the same conditions (16). Nor is there direct proportionality between cocarboxylase concentration and carbon dioxide formation. The decarboxylation of pyruvic acid is not governed by the
H
H
H
? ?
OH I
CH*-c-c-c-CH,-0-P=O
I
I 1 1
H H H
cH8-[~N;i~N\c0 CH"\
I AH
I
OH
- . - . - - specific :rotein
N//\C/
0 .
Yellow enzyme
and the yellow enzyme is a combination of this ester with a specific enzyme protein. Lactoflavin was first isolated in crystalline form from milk by Kuhn and co-workers (23) and two years later it was prepared synthetically (24). Warburg and Christian (25) obtained from red blood cells another new coenzyme and an intermediate enzyme from bottom yeast. The second coenzyme, the intermediate enzyme, and the yellow enzyine comprise the yellow enzyme system. It oxidizes hexosemonophos-
phoric acid aiirobically to phosphohexonic acid. The second coenzyme contains one molecule of adenine, one molecule of nicotinic acid amid, two molecules of pentoses (probable ribose) and three molecules of phosphoric acid. Thus i t is a triphosphopyridine nucleotide (25). The exact structure of the compound is not yet known. This coenzyme is a hydrogen transporter. It may be reversibly oxidized and reduced. The intermediate enzyme is a specific protein and is inactive by itself but is necessary for the activity of the coenzyme. The intermediate enzyme and hexosemonophosphate in the presence of a small amount of coenzyme does not react until the yellow enzyme is added.
lactoflavinmonophosphoricacid wereactiveascoenzymes in the yellow oxidation system or Warburg and Christian. The phosphoric acid ester, however, was many hundred times more active than lactoflavin. Kuhn and Rudy believe that the lactoflavinphosphate forms a h e r union with the specific enzyme protein. Lactoflavin can be separated by dialysis a t pH 7.0, whereas lactoflavinphosphoric acid is split off only when solutions of the yellow enzyme are dialyzed against HC1 (29). Thiamin, unlike lactoflavin, cannot function as coenzyme for carboxylase, nor can its monophosphoric acid ester. Only the diphosphoric acid ester is able to function as cocarboxylase.
MECHANISM OF THE YELLOW OXIDATION ENZYME SYSTEM
PHOSPHORYLATION OF LACTOFLAVIN Lactotlavin may be converted to the monophosphoric acid ester by phosphorus oxychloride (18) or by the duodenal mucosa (30). Laszt and Verzk (31) have shown that phosphorylation takes place ile &no. It may be inhibited by iodoacetate and in that case only the phosphoric acid ester of lactoflavin acts as a vitamin.
The yellow ferment is an enzyme whose specific substrates are the coenzymes (26, 27); e. g., triphosphopyridine nucleotide (of the yellow enzyme system) and diphosphopyridine nucleotide (cozymase). The hydrogen is reversibly given off by the pyridine ring of the second coenzyme and is taken up by the isoalloxazin ring of the yellow enzyme. The N-atom of position 1 and 10, respectively, each combine with one H-atom and the colorless leuco componnd forms. The two H-atoms may be removed again by oxidation and theoxidized yellow componnd is reformed: R-
R-
HsC-
COZYMASE
Cozymase, the coenzyme of "alcoholic fermentation," was discovered by Harden and Young in 1905. Shortlv after Warbure " and associates have shown that their second coenzyme is a triosephosphopyridine nucleotide, it was found in collaborative investigations by Warburg and Christian (32) and by Euler, Albers, and Schlenk (33) that nicotinic acid amid is also a part of cozymase and Euler and Schlenk (34) proposed the following structural formula for cozymase:
The mechanism of the yellow enzyme system may be expressed by the following equations:
(a) Coenzyme coenzyme
+
hexosemonophosphate = reduced
+ phosphohexonic acid (intermdiate
enzyme) (b) Reduced coenzyme yellow enzyme = coenzyme reduced yellow enzyme (6) Reduced yellow enzyme molecular oxygen (or cytochrome) = Hz0 yellow enzyme.
+
+
+
+
While the reduced yellow enzyme may be reoxidized by molecular oxygen, in the living cell the reoxidation is carried out with great speed by cytochrom C. At the 0% pressure which prevails in tissues the reduced yellow enzyme is only slightly affected by oxygen. In this reaction the yellow enzyme system functions as an oxidation or respiration system, whereas when i t transfers hydrogen to other cell constituents (acetaldehyde), via cozymase, we then speak of fermentation. The yellow enzyme system may function in the dehydrogenation of the following: hexosemonophosphate east and muscle) alcohol (yeast), glucose (liver), malic acid (muscle), lactic acid (muscle), citric acid (muscle and ~lants),hexosediphosphate (yeast and muscle) and others (26). Kuhn and Rudy (28) found that lactoihvin as well as
1
CH
I
C-N
N--C-N---
C-C-C-C-CH-&P=O
I
DIPHOSPHOPYRIDWE NUCLEOTIDE
Cozymase also is a hydrogen transporting coenzyme. By taking up hydrogen it forms dihydrocozymase. According to Euler (35) two apodehydrases are necessary for the activity of cozymase. In the alcoholic fermentation when dismutation between triosephosphoric acid and acetaldehyde occurs the following reactions take place. (a) Coz
+ triosephosphate
-
+
CoeHg phosphoglyceric acid (triosephosphate dehydrogenase)
(acetaldehyde reductase)
The reactivation of the purified liver esterase, however, requires about a thousand times more ascorbic acid than the amount present in the original enzyme preparation (one microgram per esterase unit). From this the authors conclude that similar to lactoflavin, ascorbic acid may also have to be converted to a more active form. Their hypothesis is also supported by the .,"\ fact that reactivation of the inactive enzyme by ascorJl). Cozymase had been found to be necessary in a great bic acid requires a t least forty minutes. The experinumber of enzymic reactions of which only a few can ments of Jurawicz are in harmony with the early work of be mentioned here: hexosediphosphate formation dur- Palladin (44) who fonnd blood esterase greatly deing fermentation by living yeast and by dry yeast, creased in patients suffering from scurvy and with phosphorylation by yeast phosphatese, transformation recent findings of Moster (45) who fonnd that after oral of aldehyde by yeast (cozymase = comutase), dehydro- administration of ascorbic acid the blood esterase congenation of hexosephosphate by yeast, fermentation centration greatly increased but not the lipase activity. of phosphopyruvic acid by apozymase, dehydrogena- Quastel and Wheatley (46) have shown.that fatty acid tion of lactic acid to pyruvic acid, and in the debydro- oxidation by liver tissues on the addition of ascorbic genation of other metabolites (38). acid may increase in guinea pigs up to two hundred per Cozymase appears to be an indispensable constituent cent. of almost every cell. According to Euler animals canPfleger and Scholl (47) made important observations not live without cozymase. Such a deficiency results in on diabetic patients receivingascorbicacidtogether with a general disarrangement of carbohydrate metabolism. insulin. The action of insulin was greatly increased Animals even partially deprived of cozymase die soon by the administration of ascorbic acid. It was possible without specific changes of their organs (39). Thus to reduce the insulin dosage and in all the diabetic cozymase appears to be a vitamin. patients there was an increaseof tonus and subjective improvement. Ascorbic acid alone had a favorable effecton the combustion of acetone bodies. In diabetes, Ascorbic acid, the antiscorbutic vitamin, takes part because of the restricted diet the patients suffer from in several oxidation-reduction systems (39). For in- hypovitaminoses resulting i n a more dcicctirc mctabostance, I found that peroxide-peroxidase oxidizes ascor- lism. 'I'headministration of oure vitamin Cis sueeested "" bic acid very rapidly (within a few minutes) if snb- by the authors. stances capable of forming quinones are present (41). The quinones are reduced by the ascorbic acid and they VITAMIN A AND CAROTENE (PROVITAMIN A) AS A are in turn reoxidized by peroxide-peroxidase. This COENZYME reaction results in the decomposition of the physiologically toxic peroxide. Quinone-forming compounds are Recently vitamin A has been obtained in crystalline present in plants (fivones) as well as in animal tissues form from liver oil, and i t has also been synthesized (48). (adrenaline). Besides peroxidase, ascorbic acid also It has the following structural. formula: reacts with a specific enzyme called ascorbic acid oxidase, and with phenolases (40). Cathepsin, a tissue protease, and argime (with traces of metals) increase their activity on the addition of ascorbic acid (42). Highly interesting are the observations of Jurewicz and Kraut (43) whose work with 2iuer esterase (an enzyme which hydrolyzes lower esters such as methyl acetate) indicates that ascorbic acid or probably a derivative of the vitamin functions as a coenzyme for Vitamin A this enzyme. These investigators found that certain preparations Sure, Kik, Bnchanan, and DeWitt (49) presented exof liver esterase completely lose their activity if dialyzed against dilute hydrogen chloride. The enzyme periments which showed that in vitamin A deficiency may be reactivated on the addition of ascorbic acid. there is "a marked decrease in the concentration of When hydrogen chlorideis added directly to the enzyme blood serum esterase, an appreciable decrease in hepatic solution its activity remains unchanged. Oxidation esterase, and a marked increase in hepatic lipase." Wetzler-Ligeti and Wilheim (50) made a remarkable does not alter the activity of the esterase. Guinea pigs receiving daily injections of 10-mg. doses of ascorbic observation concerning the effect of carotene, the preacid for ten days showed a considerably greater liver cursor of vitamin A, on yeast. Carotene has an acesterase concentration than the livers of the control celerating action of the fermentation of glucose by bakers' yeast. The Viennese workers have also shown animals receiving no ascorbic acid. I n vitro the yellow enzyme is able to oxidize reduced cozymase and i t probably also does so within the living cell. In alcoholic fermentation cozymase cannot be replaced by any other substance. In its functions as coenzyme for certain dehydrogenases, however, i t may be replaced by the second coenzyme of Warburg (36,
that the addition of carotene to red cells accelerates blood glycolysis. VITAMIN D-A
PHOSPHATASE REGULATOR
Many interesting papers appeared on the serum phosphatase regulating action of vitamin D as manifested in human rickets (51). The mechanism of this action is not known and these changes cannot be reproduced in experimental rickets, using rats (52, 53).
SUMMARY
Certain chemically known vitamins play important and indispensable r8les in well-defined enzymic reactions. Enzymes can be synthesized by the body. Vitamins cannot. In combination with enzymes certain vitamins catalyze the combustion of foods and the building up of new cells. This explains, in part, why lack of vitamins may result in metabolic disturbances, and in protracted and serious deficiency in death.
