- Email: [email protected]
The reaction of malic dehydrogenase with α-keto β-fluoro-succinic (β-fluoro-oxaloacetic) acid
Biochemical Pharmacology, 158, Vol. 1, pp. 207-212. Pergamon Press Ltd. Printed in Great Britain.
THE REACTION OF MALIC DEHYDROGENASE WITH a-KETO B-FLUORO-SUCCINIC (B-FLUORO-OXALOACETIC) ACID* E. KUN,f
D. R. GRASSETTI, D. W. FANSHIER and
R. M. FEATHERSTONE
Department of Pharmacology and Experimental Therapeutics, University of California Medical Center, San Francisco, California (Received 10 August 1958) Abstract-The preparation of crystalline p-fluoro-oxaloacetic acid (FOAA) from its diethyl ester is described. FOAA is a competitive inhibitor of malic dehydrogenase. MALIC dehydrogenase was obtained in pure form from pig heart by Wolfe and Neilandsl and from mitochondria of ox heart by Davies and Kun.2 A specific inhibitor of this enzyme has so far not been described. In the course of search for such an inhibitor fi-fluoro-oxaloacetic acid (FOAA) was prepared in pure form and its effect on enzymatic reactions catalyzed by malic dehydrogenase2 determined. The diethyl ester of FOAA and its Na-enolate have been previously tested for their inhibitory effects on multi-enzyme systems by Watland et aL3 and Gal,* but these forms of FOAA are not suitable for more definitive enzyme inhibition studies, The free acid has hitherto not been prepared. In this paper the preparation and properties of FOAA will be described and it will be shown that it is a competitive inhibitor of malic dehydrogenase.
EXPERIMENTAL
Preparation and properties of FOAA Diethyl fluoro-oxalacetate was prepared according to Rivett.5 It was converted to the free acid under conditions favoring transesterification.6 Hydrolysis of the ester by hydrochloric acid has been previously attempted without success.7p * The procedure used was as follows: 25 g of diethyl fluoro-oxaloacetate were dissolved in 240 ml of glacial acetic acid, then 120 ml of concentrated hydrochloric acid added. The mixture was kept at room temperature for three days, then evaporated to dryness at room temperature under reduced pressure by means of a Rinco rotating evaporator. The residue was 18.4 g of crude crystalline FOAA (85.5 per cent yield). This was purified by recrystallization from ether-petroleum ether (b.p. 30-60°C). After two recrystallizations which resulted in about 50 per cent recovery, the m.p. was 86-87” (decarboxylation) as determined with a Fisher-Johns block. Analysis: calculated for C,H,O,F: 1.5 H,O; C, 27.13; H, 3.41; F, 10.73. Found: C, 27*59; H, 3.67; F, 10.7. Neutralization equivalent: calculated 88.5; found 89.0. 2,4-dinitrophenyl-hydrazone: m.p. = 210-214” (starts decomposing between 90” and 150°C depending on the rate * Supported by grants of the U.S. Public Health Service (CY-3861, C-321 1, and H-2897). t Established Investigator of the American Heart Association, Inc. 207
208
E.
KUN, D. R. GRASSETTI,D. W. FANSHIERand R. M. FEATHER~TONE
of heating). Calculated for C~~H~N~O~F: C, 36.37; H, Z-14; N, 16.97. Found: C, 36.6; H, 2.3 ; N, 16.9. This derivative is very hygroscopic. An aqueous solution of FOAA gives a deep purple-red color with aqueous FeCI,. The color reaches its maximum intensity after about ten minutes. The crystalline acid is stable for about a month at room temperature, then slowly decarboxylates. FOAA is extremely soluble in water. Analysis and neutralization equivalent show that FOAA contains l-5 moles of H,O. Spectral evidence indicates that the keto group is in the hydrated form. This would be expected from the strongly electron attracting properties of fluorine which increase the positive character of the neighboring carbon atoms. The UV spectra of aqueous soIutions of OAA and FOAA were determined at pH l-5 (OAA, E* = 612; FOAA, E = 96) and at pH 11 (OAA, E = 798; FOAA, E = 246) at h,%, = 265 rnp for OAA and Xma, = 270 rnp for FOAA. The UV absorption spectra of both acids are shown in Fig. 1. It is apparent that in aqueous solutions FOAA is not appreciably enolized. This is further confirmed by infrared (IR) spectra, which are shown in Fig. 2. Examination of the IR spectra shows that: (1) FOAA does not exhibit the strong band at 6.1 p, which is given by OAA and has been attributed to the resonance-stabilized chelated enol.gt The hydrated form could not give an enolic chelate. The changes in the OH region are consistent with this view. (2) FOAA has only one carbonyl band in the 5 ILregion (carboxyl), whereas OAA has two bands in this region (keto group and carboxyl). (3) FOAA has a band at 9.14 p, due to the C-F bond,8 which is absent in OAA. Preparation of malic dehydrogenase Malic dehydrogenase of rat liver mitochondria was prepared as described. by Davies and Kun,2 except the enzyme was not purified beyond the second stage. In calculating turnover numbers and expressing the amounts of enzyme, the molecular
‘Etr
A - %_ )(
Mol. I.~--wt.
‘rThis is due to a carbonyl in which the double bond character has been weakened by resonance
as follows.
This is not pcxsiblc in HO-C---C-C-~-OH ri_ / OH---O
(hydrated form of FOAA)
The reaction of malic dehydrogenase
with a-keto -pfluorosuccinic
230
300
(p-fluoro-oxaloacetic)
acid 209
350
FOG. 1. UV. absorption spectra of OAA and FOAA at pH I.5 and 11.0. OAA = 16.4mg per cent, FOAA = 14.0 mg per cent. Readings were made with the Beckman DU spectraphotometer.
OAA-
FIG. 2. I.R. spectra of FAA and FOAA were made with the PERKS-ELMER
21 instrument
in
KBr disks. weight of 15,000 was used for mitochondrial malic dehydrogenase.2 Conditions of enzyme assay as well as sp~i~cation of substrates were the same as previously described.2 Determinations of Kr’were carried out by the method of Dixon.lO
The effect of FOAA on the reduction of OAA to malic acid by DPNH in the presence of malic dehydrogenase is shown in Fig. 3. In the presence of 20 pmoles of OAA per 3 ml reaction mixture, an approximately equal amount of FOAA (23.5
210
E.
KUN,
D. R.
GRASSETTI.
D. W.
FANSHIER and
R. M.
FEATHERSTONE
FIG. 3. The effect of FOAA on the reduction of OAAbyDPNHin thepresenceof malicdehydrogenase. pH = 6.8; OAA = 20 moles; FOAA = 23.5 moles; enzyme = 0.3 x 10-a pmoles; versene 69 pmoles; volume = 3.0 ml. All reductions of OAA and FOAA were made in 0.5 M PO1 buffer.
I
I
0
5
M
IO
FOAA
XlO-4
15
20
FIG. 4. The reduction of FOAA by DPNH in presence of dehydrogenase. DPNH = 0.28 pmoles. Enzyme = 0.72 x 10m3rmoles; pH = 6.8; versene = 69 rmoles volume = 3.0 ml.
caused marked inhibition. It was noticed that a slow but definite rate of DPNH oxidation occurred in the presence of FOAA alone. This reaction was studied in the presence of high enzyme concentration and it was clearly shown that FOAA is enzymatically reduced to fluoromalate. The relationship between FOAA concentration and reaction rate is shown in Fig. 4. Km for FOAA was found to be 1.3 x 1O-4 M. While the turnover number for OAA is 12,200,2 for FOAA it is between 2 and 3. These observations suggest that FOAA is a competitive inhibitor of mitochondrial malic dehydrogenase. Kinetic measurements substantiated this prediction. Competition between OAA and FOAA is shown in Fig. 5, which is a typical Dixon type plot.lO The Ki value of 2.8 x 1O-5 was determined by this graphical method.
pmoles)
Since Km for OAA was known2 1. 1 the v axts at V
i
could be calculated. IniLX
The horizontal
line drawn
of the lines constructed _meets the point of intersection max from rate values at two OAA concentrations. The inhibition of malate oxidation by FOAA is shown in Fig. 6. The value of Ki of 7.4 x 1O-6 was determined graphically from reaction velocity data obtained at various through
The reaction of ma&c dehydrogenase
with a-keto ~-~uorosuccinic
~~-fluoro-oxaloa~t~c)
acid
21 I
lNHl8ITlON OF OAA+MALATE REACTION BY WAA
FOG. 5. Competition
between OAA and FOAA. Enzyme = 0.35 x 1W3 @moles; DPNH = 028 pmoles. INHIBiTlON OF MALATE-OAA REACTION BY FOAA
Fm. 6, Inhibition of malate oxidation by FOAA; malate = 50 pmoles; DPN = I-5 ~moles; Tris buffer @1 M, pH 8.4; enzyme = 0.38 x 10" ,umoles; volume = 3.0 ml,
FOAA ~o~ce~trat~ons and from known values of Km for mafate (between 5 x to-* to lO-a M).2 Similar competitive type of inhibition was obtained with other substrates of mitochondrial malic dehydrogenase such as meso-tartrate (Fig. 7) and dioxosuccinate. The extreme instability of dioxosuccinate made kinetic measurements somewhat uncertain and the KI value of 1.8 x 1O-6 is a close approximation for this substrate, lN~iBlT{ON OF MESOTARTRATE-+DWF REACTiON EJY FOAA
FE. 7. Inhibition
of oxidation of Mao-titrate by FOAA. Mesotartrate = 150 ~rnol~;~PN pmoles; Tris buffer 0.1 M, pH 8.4; enzyme = I.4 x 10-3~moles,
= 1.5
212
E. KUN, D. R. GRASSETTI, D. W. FANSHIERand R. M. FEATHERSTONE DISCUSSION
The simplest interpretation of the kinetic data is that FOAA inhibits malic dehydrogenase by combining with the enzyme at the site of OAA. The introduction of the fluorine atom into OAA increases its acid strength* and markedly reduces enolization, as shown by UV and IR spectra. These two effects may well contribute to the inhibitory action of FOAA. Since an increase in FOAA concentration results in progressive increase in inhibition at fixed levels of substrate, this inhibition appears to be of the fully competitive type (Dixon and Webb).ll It is interesting that Ki values for OAA, mesotartrate and dioxosuccinate are of the same order of magnitude (between 2 to 3 x 10-5). This fact supports the view proposed by Davies and Kun2 that malic dehydrogenase reacts with all these substrates. It is predictable from the lower Ki value for L(+) malate (7.4 x lO-‘j) that the reaction from malate to OAA is inhibited by FOAA to a greater extent than the reverse. It is to be expected that in a multienzyme system in which malate is formed, FOAA would cause the channeling of malate metabolism in directions other than oxidation to OAA. FOAA is a slowly reacting substrate for malic dehydrogenase. The product, fluoromalate, was shown by Krebs and Davies12 and by Gal4 to inhibit the malic enzyme. From the inhibitory properties of FOAA it appears likely that pharmacological effects of this compound would not show up in the form of acute toxicity since an alteration rather than inhibition of the tricarboxylic acid cycle is the result. Investigations dealing with the metabolic effects of chronic FOAA feeding as well as reactions of FOAA with other enzymes are in progress. * OAA pk,’ = 2.7 pk,’ = 4.1
FOAA
$11 z ;.; 2 .
Acknowledgemenr-It is a pleasure to acknowledge the contribution of Mr. M. K. Hrenoff from the Spectrographic Laboratory of the University of California Medical Center for the preparation of infra-red spectra.
REFERENCES 1. R. G. WOLFE and J. B. NEILANDS, J. Biol. Chem. 221, 61 (1956). 2. D. D. DAVIES and E. KUN, Biochem. J. 66, 307 (1957). 3. D. E. WATLAND, S. C. WANG, G. KALNITSKY and J. P. H~MMEL, Arch. Biochem. Biophys. 67, 138 (1957). 4. E. M. GAL, Arch. Biochem. Biophys. 73, 279 (1958). 5. D. E. A. RIVE-IT, J. Chem. Sot. 3710 (1953). 6. M. A. MITZ, A. E. AXELROD, K. HOFMANN, J. Amer. Chem. Sot. 72, 1231 (1950). 7. I. BLANK and J. MAGER, Experientia 10,77 (1954). 8. I. BLANK, J. MAGER and E. D. BERGMANN,J. Chem. Sot. 2190 (1955). 9. L. J. BELLAMY, The Infrared Spectra of Complex Molecules, p. 157. Wiley, New York (1954). 10. M. DIXON, Biochem. J. 55, 170 (1953). 11. M. DIXON and E. C. WEBB, Enzymes, p. 172. Longmans, Green, London (1958). 12. H. A. KREBS and D. D. DAVIES, Arch. Sci. Biol. (Napoli) 39, 533 (1955).
THE REACTION OF MALIC DEHYDROGENASE WITH a-KETO B-FLUORO-SUCCINIC (B-FLUORO-OXALOACETIC) ACID* E. KUN,f
D. R. GRASSETTI, D. W. FANSHIER and
R. M. FEATHERSTONE
Department of Pharmacology and Experimental Therapeutics, University of California Medical Center, San Francisco, California (Received 10 August 1958) Abstract-The preparation of crystalline p-fluoro-oxaloacetic acid (FOAA) from its diethyl ester is described. FOAA is a competitive inhibitor of malic dehydrogenase. MALIC dehydrogenase was obtained in pure form from pig heart by Wolfe and Neilandsl and from mitochondria of ox heart by Davies and Kun.2 A specific inhibitor of this enzyme has so far not been described. In the course of search for such an inhibitor fi-fluoro-oxaloacetic acid (FOAA) was prepared in pure form and its effect on enzymatic reactions catalyzed by malic dehydrogenase2 determined. The diethyl ester of FOAA and its Na-enolate have been previously tested for their inhibitory effects on multi-enzyme systems by Watland et aL3 and Gal,* but these forms of FOAA are not suitable for more definitive enzyme inhibition studies, The free acid has hitherto not been prepared. In this paper the preparation and properties of FOAA will be described and it will be shown that it is a competitive inhibitor of malic dehydrogenase.
EXPERIMENTAL
Preparation and properties of FOAA Diethyl fluoro-oxalacetate was prepared according to Rivett.5 It was converted to the free acid under conditions favoring transesterification.6 Hydrolysis of the ester by hydrochloric acid has been previously attempted without success.7p * The procedure used was as follows: 25 g of diethyl fluoro-oxaloacetate were dissolved in 240 ml of glacial acetic acid, then 120 ml of concentrated hydrochloric acid added. The mixture was kept at room temperature for three days, then evaporated to dryness at room temperature under reduced pressure by means of a Rinco rotating evaporator. The residue was 18.4 g of crude crystalline FOAA (85.5 per cent yield). This was purified by recrystallization from ether-petroleum ether (b.p. 30-60°C). After two recrystallizations which resulted in about 50 per cent recovery, the m.p. was 86-87” (decarboxylation) as determined with a Fisher-Johns block. Analysis: calculated for C,H,O,F: 1.5 H,O; C, 27.13; H, 3.41; F, 10.73. Found: C, 27*59; H, 3.67; F, 10.7. Neutralization equivalent: calculated 88.5; found 89.0. 2,4-dinitrophenyl-hydrazone: m.p. = 210-214” (starts decomposing between 90” and 150°C depending on the rate * Supported by grants of the U.S. Public Health Service (CY-3861, C-321 1, and H-2897). t Established Investigator of the American Heart Association, Inc. 207
208
E.
KUN, D. R. GRASSETTI,D. W. FANSHIERand R. M. FEATHER~TONE
of heating). Calculated for C~~H~N~O~F: C, 36.37; H, Z-14; N, 16.97. Found: C, 36.6; H, 2.3 ; N, 16.9. This derivative is very hygroscopic. An aqueous solution of FOAA gives a deep purple-red color with aqueous FeCI,. The color reaches its maximum intensity after about ten minutes. The crystalline acid is stable for about a month at room temperature, then slowly decarboxylates. FOAA is extremely soluble in water. Analysis and neutralization equivalent show that FOAA contains l-5 moles of H,O. Spectral evidence indicates that the keto group is in the hydrated form. This would be expected from the strongly electron attracting properties of fluorine which increase the positive character of the neighboring carbon atoms. The UV spectra of aqueous soIutions of OAA and FOAA were determined at pH l-5 (OAA, E* = 612; FOAA, E = 96) and at pH 11 (OAA, E = 798; FOAA, E = 246) at h,%, = 265 rnp for OAA and Xma, = 270 rnp for FOAA. The UV absorption spectra of both acids are shown in Fig. 1. It is apparent that in aqueous solutions FOAA is not appreciably enolized. This is further confirmed by infrared (IR) spectra, which are shown in Fig. 2. Examination of the IR spectra shows that: (1) FOAA does not exhibit the strong band at 6.1 p, which is given by OAA and has been attributed to the resonance-stabilized chelated enol.gt The hydrated form could not give an enolic chelate. The changes in the OH region are consistent with this view. (2) FOAA has only one carbonyl band in the 5 ILregion (carboxyl), whereas OAA has two bands in this region (keto group and carboxyl). (3) FOAA has a band at 9.14 p, due to the C-F bond,8 which is absent in OAA. Preparation of malic dehydrogenase Malic dehydrogenase of rat liver mitochondria was prepared as described. by Davies and Kun,2 except the enzyme was not purified beyond the second stage. In calculating turnover numbers and expressing the amounts of enzyme, the molecular
‘Etr
A - %_ )(
Mol. I.~--wt.
‘rThis is due to a carbonyl in which the double bond character has been weakened by resonance
as follows.
This is not pcxsiblc in HO-C---C-C-~-OH ri_ / OH---O
(hydrated form of FOAA)
The reaction of malic dehydrogenase
with a-keto -pfluorosuccinic
230
300
(p-fluoro-oxaloacetic)
acid 209
350
FOG. 1. UV. absorption spectra of OAA and FOAA at pH I.5 and 11.0. OAA = 16.4mg per cent, FOAA = 14.0 mg per cent. Readings were made with the Beckman DU spectraphotometer.
OAA-
FIG. 2. I.R. spectra of FAA and FOAA were made with the PERKS-ELMER
21 instrument
in
KBr disks. weight of 15,000 was used for mitochondrial malic dehydrogenase.2 Conditions of enzyme assay as well as sp~i~cation of substrates were the same as previously described.2 Determinations of Kr’were carried out by the method of Dixon.lO
The effect of FOAA on the reduction of OAA to malic acid by DPNH in the presence of malic dehydrogenase is shown in Fig. 3. In the presence of 20 pmoles of OAA per 3 ml reaction mixture, an approximately equal amount of FOAA (23.5
210
E.
KUN,
D. R.
GRASSETTI.
D. W.
FANSHIER and
R. M.
FEATHERSTONE
FIG. 3. The effect of FOAA on the reduction of OAAbyDPNHin thepresenceof malicdehydrogenase. pH = 6.8; OAA = 20 moles; FOAA = 23.5 moles; enzyme = 0.3 x 10-a pmoles; versene 69 pmoles; volume = 3.0 ml. All reductions of OAA and FOAA were made in 0.5 M PO1 buffer.
I
I
0
5
M
IO
FOAA
XlO-4
15
20
FIG. 4. The reduction of FOAA by DPNH in presence of dehydrogenase. DPNH = 0.28 pmoles. Enzyme = 0.72 x 10m3rmoles; pH = 6.8; versene = 69 rmoles volume = 3.0 ml.
caused marked inhibition. It was noticed that a slow but definite rate of DPNH oxidation occurred in the presence of FOAA alone. This reaction was studied in the presence of high enzyme concentration and it was clearly shown that FOAA is enzymatically reduced to fluoromalate. The relationship between FOAA concentration and reaction rate is shown in Fig. 4. Km for FOAA was found to be 1.3 x 1O-4 M. While the turnover number for OAA is 12,200,2 for FOAA it is between 2 and 3. These observations suggest that FOAA is a competitive inhibitor of mitochondrial malic dehydrogenase. Kinetic measurements substantiated this prediction. Competition between OAA and FOAA is shown in Fig. 5, which is a typical Dixon type plot.lO The Ki value of 2.8 x 1O-5 was determined by this graphical method.
pmoles)
Since Km for OAA was known2 1. 1 the v axts at V
i
could be calculated. IniLX
The horizontal
line drawn
of the lines constructed _meets the point of intersection max from rate values at two OAA concentrations. The inhibition of malate oxidation by FOAA is shown in Fig. 6. The value of Ki of 7.4 x 1O-6 was determined graphically from reaction velocity data obtained at various through
The reaction of ma&c dehydrogenase
with a-keto ~-~uorosuccinic
~~-fluoro-oxaloa~t~c)
acid
21 I
lNHl8ITlON OF OAA+MALATE REACTION BY WAA
FOG. 5. Competition
between OAA and FOAA. Enzyme = 0.35 x 1W3 @moles; DPNH = 028 pmoles. INHIBiTlON OF MALATE-OAA REACTION BY FOAA
Fm. 6, Inhibition of malate oxidation by FOAA; malate = 50 pmoles; DPN = I-5 ~moles; Tris buffer @1 M, pH 8.4; enzyme = 0.38 x 10" ,umoles; volume = 3.0 ml,
FOAA ~o~ce~trat~ons and from known values of Km for mafate (between 5 x to-* to lO-a M).2 Similar competitive type of inhibition was obtained with other substrates of mitochondrial malic dehydrogenase such as meso-tartrate (Fig. 7) and dioxosuccinate. The extreme instability of dioxosuccinate made kinetic measurements somewhat uncertain and the KI value of 1.8 x 1O-6 is a close approximation for this substrate, lN~iBlT{ON OF MESOTARTRATE-+DWF REACTiON EJY FOAA
FE. 7. Inhibition
of oxidation of Mao-titrate by FOAA. Mesotartrate = 150 ~rnol~;~PN pmoles; Tris buffer 0.1 M, pH 8.4; enzyme = I.4 x 10-3~moles,
= 1.5
212
E. KUN, D. R. GRASSETTI, D. W. FANSHIERand R. M. FEATHERSTONE DISCUSSION
The simplest interpretation of the kinetic data is that FOAA inhibits malic dehydrogenase by combining with the enzyme at the site of OAA. The introduction of the fluorine atom into OAA increases its acid strength* and markedly reduces enolization, as shown by UV and IR spectra. These two effects may well contribute to the inhibitory action of FOAA. Since an increase in FOAA concentration results in progressive increase in inhibition at fixed levels of substrate, this inhibition appears to be of the fully competitive type (Dixon and Webb).ll It is interesting that Ki values for OAA, mesotartrate and dioxosuccinate are of the same order of magnitude (between 2 to 3 x 10-5). This fact supports the view proposed by Davies and Kun2 that malic dehydrogenase reacts with all these substrates. It is predictable from the lower Ki value for L(+) malate (7.4 x lO-‘j) that the reaction from malate to OAA is inhibited by FOAA to a greater extent than the reverse. It is to be expected that in a multienzyme system in which malate is formed, FOAA would cause the channeling of malate metabolism in directions other than oxidation to OAA. FOAA is a slowly reacting substrate for malic dehydrogenase. The product, fluoromalate, was shown by Krebs and Davies12 and by Gal4 to inhibit the malic enzyme. From the inhibitory properties of FOAA it appears likely that pharmacological effects of this compound would not show up in the form of acute toxicity since an alteration rather than inhibition of the tricarboxylic acid cycle is the result. Investigations dealing with the metabolic effects of chronic FOAA feeding as well as reactions of FOAA with other enzymes are in progress. * OAA pk,’ = 2.7 pk,’ = 4.1
FOAA
$11 z ;.; 2 .
Acknowledgemenr-It is a pleasure to acknowledge the contribution of Mr. M. K. Hrenoff from the Spectrographic Laboratory of the University of California Medical Center for the preparation of infra-red spectra.
REFERENCES 1. R. G. WOLFE and J. B. NEILANDS, J. Biol. Chem. 221, 61 (1956). 2. D. D. DAVIES and E. KUN, Biochem. J. 66, 307 (1957). 3. D. E. WATLAND, S. C. WANG, G. KALNITSKY and J. P. H~MMEL, Arch. Biochem. Biophys. 67, 138 (1957). 4. E. M. GAL, Arch. Biochem. Biophys. 73, 279 (1958). 5. D. E. A. RIVE-IT, J. Chem. Sot. 3710 (1953). 6. M. A. MITZ, A. E. AXELROD, K. HOFMANN, J. Amer. Chem. Sot. 72, 1231 (1950). 7. I. BLANK and J. MAGER, Experientia 10,77 (1954). 8. I. BLANK, J. MAGER and E. D. BERGMANN,J. Chem. Sot. 2190 (1955). 9. L. J. BELLAMY, The Infrared Spectra of Complex Molecules, p. 157. Wiley, New York (1954). 10. M. DIXON, Biochem. J. 55, 170 (1953). 11. M. DIXON and E. C. WEBB, Enzymes, p. 172. Longmans, Green, London (1958). 12. H. A. KREBS and D. D. DAVIES, Arch. Sci. Biol. (Napoli) 39, 533 (1955).