ARTICLE:
ENZYMATIC MECHANISM OF
CREATINE SYNTHESIS
G. L. Cantoni and P. J. Vignos, Jr.
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J. Biol. Chem. 1954, 209:647-659.
ENZYMATIC
MECHANISM
BY G. L. CANTON1
(From
the Department
OF CREATINE
AND
P. J. VIGNOS,
of Pharmacology,
School of Medicine,
University,
Cleveland,
Ohio)
(Received
for
publication,
March
SYNTHESIS*
JR.t
Western
Reserve
1, 1954)
* Fifth
paper of a series on enzymatic
mechanisms
in transmethylation.
This investigation
was supported
in part
by grants-in-aid
from
the Williams-Waterman
Fund for the Combat
of Dietary
Diseases
of the Research
Corporation
of New York
and from the American
Cancer
Society.
Presented
in part at the Forty-third
annual meeting
of the American
Society
of Biological
Chemists,
New York,
April,
1952 (1).
t Fellow
of the United
States Public
Health
Service.
Present
address,
Department
of Medicine,
School
of Medicine,
Western
Reserve
University,
Cleveland,
Ohio.
1 The following
abbreviations
are used: GA, guanidinoacetic
acid or guanidinoacetate;
AMe,
S-adenosylmethionine,
i.e. active
methionine;
ASR, adenosylhomocysteine;
NMeN,
Ni-methylnicotinamide;
ATP,
adenosinetriphosphate;
ADP,
adenosinediphosphate;
GSH,
reduced
glutathione;
IP, orthophosphate;
Tris,
tris(hydroxymethyl)aminomethane.
647
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It is well established that the last step in the biosynthesis of creatine
involves the methylation of guanidinoacetic acid.’ This conclusion is
based upon experimental evidence derived from two independent lines of
investigation. By application of the isotopic tracer technique, du Vigneaud
et al. (2) have demonstrated that the methyl group in creatine is derived
from L-methionine; furthermore, these authors obtained conclusive evidence that, in wivo, the methyl group of L-methionine is transferred to the
methyl acceptor as a unit. In an independent study of this transmethylation reaction in vitro, Borsook and Dubnoff (3) reached similar conclusions
using guinea pig liver slices. Subsequently (4), these authors have shown
that cell-free liver homogenates fortified with adenylic acid and an oxidizable substrate such as a-ketoglutaric acid are able to form creatine under
aerobic conditions. It was assumed by these authors and by others
(5, 6) that these requirements were a reflection of the endergonic nature
of this transmethylation reaction and an indication of the ability of ATP
to serve as an energy source in this system. These conclusions appeared
to have been borne out by the findings of Cohen (5) that the methylation
of guanidinoacetic acid proceeds anaerobically in the presence of ATP
and Mg++.
The biosyntheses of creatine and W-methylnicotinamide
are similar.
In both casesthe methyl group is derived from L-methionine and, furthermore, ATP and Mg++ are required. Recent investigations (6, 7) have
648
CREATINE
SYNTHESIS
clarified the enzymatic mechanisms involved in the biosynthesis of NMeN
and have indicated that this transmethylation
reaction proceeds in a stepwise fashion according to Reactions 1 and 2 which, respectively,
are catalyzed by the methionine-activating
enzyme and by nicotinamide methylpherase.
Mg++, GSH
(1) r,-Methionine
+ ATP
(2) AMe + nicotinsmide + NMeN
(1) + (2), L-methionine
+ S-adenosylmethionine
+ S-adenosylhomocysteine
+ ATP + nicotinamide
+ NMeN
+ 31P
+ 31P + ASR
(3’)
(1) + (3), L-methionine
AMe + GA +
creatine
+ ATP + GA M
+ ASR + H+
GSH
Mg++
creatine
+ ASR + H+ + 31P
Preliminary experiments gave indications that, indeed, creatine synthesis followed this pattern, and it was considered of interest to study the
synthesis of creatine from GA and AMe in more detail. A soluble enzyme2 which catalyzes Reaction 3 has been found in cell-free extracts of
guinea pig, rabbit, beef, and pig liver. The enzyme from pig liver has
been purified approximately 20-fold by means of ammonium sulfate fractionation followed by treatment with alumina Gy. The partially purified
enzyme is free of methionine-activating
enzyme and of nicotinamide
methylpherase. Glutathione or other reducing substances are required
for the optimal activity of the enzyme. No evidence*has yet been obtained to indicate the participation of metal ions or other cofactors in the
reaction.
Greatine was conclusively identified as one of the products of the enzymatic reaction by (a) the close agreement in its chemical determination
by two different methods, namely, the a-naphthol-diacetyl reaction and
the Jaffe alkaline picrate test, (b) the ability of the reaction product to
z By analogy with the nomenclature
adopted in earlier studies of this series, the
enzyme oatalyzing Reaction 3 will be referred to ae guanidinoacetate
methylpheraze
(GA methylpheraee).
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It has been estimated (8) that the methylsulfonium bond in AMe and
other sulfonium compounds is energy-rich and, tentatively, it has been
assumed that the onium bond energy might account for the biological
lability of the methyl group in AMe.
It has been suggested (8) that the activation of methionine might be a
prerequisite
to the transfer of its methyl group to any one of a variety of
methyl acceptors. According to this hypothesis the biosynthesis of creatine from r,-methionine, ATP, and GA should involve the coupling of Reaction 1 with Reaction 3.
Q.
L.
CANTONI
AND
P.
J.
VIQNOS,
JR.
649
function as a substrate for creatine kinase, an enzyme which catalyzes
Reaction 4
Creatine + ATP = creatine phosphate + ADP + H+
(4)
and (c) the isolation of creatinine as the potassium picrate double salt
from the protein-free titrate obtained in a large scale enzymatic run.
EXPERIMENTAL
Enzyme Preparations
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GA Methylpheraae-GA
methylpheraae was found in cell-free extracta
of rabbit, guinea pig, beef, and pig livers. Pig liver extra& were selected
for purification.
For preparation of the enzyme fresh pig liver was obtained at the slaughter-house, packed in ice, and brought to the laboratory
in a vacuum container.
All the manipulations
were carried out in a cold
room maintained at 2”. The purification may be interrupted after each
ammonium sulfate fractionation and the preparation can be stored at -20”
as an ammonium sulfate paste. The liver was diced, rinsed free of excess
blood with a buffer solution (sodium acetate 0.075 M, pH 5.0), weighed,
and homogenized in a Waring blendor with 2.5 volumes of the same buffer
solution.
Next the homogenate was centrifuged at 9000 r.p.m. for 30
minutes.
The supernatant material, which was slightly opalescent, was
packed in ice and solid ammonium sulfate was added slowly with mechanical stirring (19.5 gm. per 100 ml.). The precipitate was removed by centrifugation
in a Servall high speed centrifuge and discarded, and ammonium sulfate (10.5 gm. per 100 ml.) was added to the supernatant
solution.
The precipitate collected as above contained essentially all
of the activity.
For further purification the ammonium
sulfate paste
was dissolved in a small volume of 0.10 M sodium acetate and dialyzed for
3 hours against running 0.05 M acetate buffer, pH 5.6. At the end of
the dialysis an inactive precipitate was removed by centrifugation.
The
protein content of the supernatant material was then determined, and the
protein concentration was adjusted, by dilution with the same acetate buffer, to 20 mg. per ml.; 0.33 volume of alumina 0y (dry weight, 35 mg. per
ml.) was added, with good mechanical stirring, the suspension was centrifuged at 3000 r.p.m., and the supernatant fluid discarded. The residue
was eluted four times with phosphate buffer (0.0125 M, pH 6.35), a volume
of buffer equal to that of the alumina Ov suspension being used each time.
The eluates having the highest specific activity, usually the fist two, were
pooled, and the pH of the solution was adjusted to 7.2 with dilute NaOH,
and then buffered at this pH by addition of 0.05 volume of 2 M phosphate
buffer, pH 7.2. Next, saturated ammonium sulfate, pH 7.2, was added
to 47.5 per cent saturation and the inert precipitat.e removed at high speed
650
CREATINE
SYNTHESIS
centrifugation
as above. Solid ammonium sulfate was added slowly with
stirring to the supernatant
solution (1 gm. for each 10 ml.) and, after 30
minutes, the precipitate was collected by centrifugation.
The precipitate
was dissolved in dilute phosphate buffer (pH 7.4) and, if convenient, dialyzed against 0.05 M KC1 or 0.025 M phosphate buffer (pH 7.4) for 3 hours.
The results from a representative
run are presented in Table I.
Other Enzyme Preparations-Creatine
kinase was prepared from a water
(2.5 volumes) extract of rabbit muscle.
The muscle extract was dialyzed
against, running distilled water for 12 hours in the cold. A heavy flocculent, precipitate formed and was discarded and the supernatant material
was fractionated by ammonium sulfate. The fraction which precipitated
of Guanidinoacetate
I
Methylpherase
from Pig Liver
-
Units per ml.’
Specificactivity
.Y
Acetate btierextract.............
Ammonium Sulfate Ppt. I (2540%
saturated)....................
Treatment with alumina &
Supernatant....................
Eluatel........................
‘I
2........................
“
3. . . . . . . . . . . . . . . . . . .
Ammonium Sulfate Ppt . II (48-6570
saturated)....................
1.1
nils per ma. pmlcilII
0.034
per ccn:
100
3.2
0.13
90
0.56
2.76
2.38
0.83
0.14
1.6
1.7
1.66
19.5
28.7
25.6
9
15.6
2.6
-
* 1 unit - 1 PM of creatine
Yield
_-
formed in 120 minutes
34.5
-
at 37”.
between 60 and 70 per cent saturationa was collected and dissolved in cold
0.85 M NaCl. The ATPase activity of this fraction was very slight, and
could be reduced to insignificantvalues by dilution.
The preparations of nicotinamide methylpherase and of methionineactivating enzyme were as described earlier (7, 9).
Chemical Preparations-S-Adenosylmethionine
was prepared enzymatically and purified as described by Cantoni (9). Unless indicated the
preparation
of AMe contained L-methionine, but was free of organic and
inorganic
phosphate
compounds
and of Mg++. Preparations of AMe,
free of methionine and approximately 80 per cent pure (AMe SO), obtained
by paper
chromatography
were
used in some of the experiments.
Guan-
idinoacetic acid obtained commercially was recrystallized from water
before use. Reduced glutathione and ATP were commercial preparations.
* 60 per cent saturation
= 42.3 gm. per 100 ml.
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TABLE
Preparation
0.
L.
CANTONI
AND
P.
J.
VIGNOS,
651
JR.
p-Chloromercuribenaoic
acid and methionine methylsulfonium
iodide were
generously supplied by Dr. T. Singer and Dr. R. McRorie.
Alumina Ou
was prepared as described by Bauer (10).
Measurement of Enzyme Activity-The
reaction was carried out in small
test-tubes.
The cold enzyme solution was added to the reaction mixture
at room temperature and the reaction run for 60 to 120 minutes in a water
bath at 37”. Under the conditions of the assay the activity of the enzyme
was linear with time and proportional to enzyme concentration (Fig. 1).
t?
W
z
%
.25 -
I?
E
*
.I25 -
0.1
ml ENZYME
FIG.
1. Relationship
I
0.2
/ml R.M.
of enzyme concentration
t 3
to activity
After stopping the reaction by the addition of trichloroacetic acid, an
aliquot of the protein-free filtrate was autoclaved in 0.5 N HCl for 30 minutes at 15 pounds pressure and the resulting creatine determined by Borsook’s modification
(11) of the alkaline picrate method of Folin (12).
The amount of creatine formed was determined by the difference between the color developed in the complete system and that developed
in a duplicate sample in which AMe had been added after the completion
of the incubation.
This procedure was aimed at correcting for any preformed creatine, as well as for any chromogenic material derived from
guanidinoacetic acid. In addition, a variety of control experiments were
performed to make sure that the increase in creatinine was in reality due
to creatine synthesis and not to the formation of chromogenic “creatine”-
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i
.5d
-z
.
f3
z ,375 -
652
CREATINE
SYNTHESIS
like substances derived from AMe, GA, or the enzyme preparation itself.
Under the experimental condition used there was no detectable increase in
chromogenic material unless all of the components of the reaction mixture
were added (Table II).
Specificity and Properties of GA Methylpherase-It
was found that the
activity of GA methylpherase is a function of the concentration of the
substrate both with regard to GA and to AMe. In the presence of an
excess of the acceptor, GA; the transfer of the methyl group of S-adenosylmethionine appears to go to completion as indicated by the stoichiometric
relationship between the amount of substrate furnished and the amount
of creatine formed. Likewise, in the presence of an excess of the methyl
Methylation
II
of Guanidinoacetate
Components
of system,
0.15 ml. of guanidinoacetate methylpherase (Cy eluate
pool containing
1.75 mg. of protein) in a final volume of 0.8 ml. The complete system contained
GA, 0.0033 M; AMe,
0.0021 M; BAL,
0.00016
M; and Tris buffer,
pH
7.4,0.1
M. Incubation
time, 60 minutes
at 37”.
The results
are expressed
as micromoles of creatine
formed
per ml. of enzyme
preparation
per hour.
I
Complete
system .....................
No GA. ..............................
1t AMe ..............................
“ enzyme.
..........................
‘I BAL ..............................
Creatine formed
1.10
0
0
0
0.605
Per cent of complete system
100
0
0
0
55
donor, all of the guanidinoacetate supplied can be methylated to creatine
(Table III).
A number of S-methyl compounds related chemically to AMe, such as
adeninethiomethylpentose,
and methionine methylsulfonium
iodide were
tested for their ability to function as methyl donors in this system; only
AMe was active as a methyl donor. L-Methionine
was not active as a
methyl donor, either in the presence or in the absence of ATP and Mg++.
However, when the system was supplemented with a preparation of methionine-activating
enzyme from rabbit liver, creatine synthesis was readily
achieved, thus providing excellent support for the reaction mechanism
described in Reactions 1 and 3 (Table IV).
The specificity of the enzyme for the methyl acceptor has been tested
only with respect to nicotinamide;
this compound was not methylated
in this system.
The pH optimum for the reaction was found to be around 7.5; phosphate, bicarbonate-COz,
and tris(hydroxymethyl)aminomethane
can be
used to buffer the reaction mixture.
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TABLE
Enzymatic
G.
L.
CANTONI
AND
P.
J.
VIGNOS,
653
JR.
TABLE
III
of Guanidinoacetic
Acid and S-Adenosylmethionine
to Amount of Creatine Formed
Equivalence
Utilization
0.3 ml. of enzyme in u final volume of 1.0 ml. Tris buffer, 0.1 M, pH 7.5; BAT,,
0.0002 M; AMe 80 and GA added aa micromoles per ml. of reaction mixture as indicated. Incubation
time, 180 minutes at 37“. The results are expressed as micromoles of creatine formed per ml. of reaction mixture.
I
Experiment
A*
Additions
GA
Experiment
Additions
ye&tiI$
AMe
80
Bt
Creatine
A&
80
formed
GA
0.085
0.17
0.26
0.34
0.42
* Enzyme (Ammonium
t Enzyme (Ammonium
0.084
0.169
0.284
0.351
0.423
2.5
2.5
2.5
2.5
2.5
2.5
2.5
0.2
0.36
0.48
0.6
0.9
1.2
0.17
0.31
0.44
0.53
0.73
0.96
84.0
87.2
91.2
89.0
82.0
80.0
Sulfate II) containing 9.1 mg. of protein per ml.
Sulfate I) containing 60.4 mg. of protein per ml.
TABLE
IV
Synthesis of Creatine by Coupling of Methionine-Activating
Enzyme
and GA Methylpherase
The reaction mixture contains ATP, 0.013 M; L-methionine,
0.02 M; GSH, 0.001
M; MgCl, 0.166 M; Tris buffer, pH 7.4,0.075 M; Enzyme 1 and Enzyme 2 as indicated.
Enzyme 1 (methionine-activating
enzyme) contains 22 mg. of protein per ml.; Enzyme 2 (guanidinoacetate
methylpheraee)
contains 6.0 mg. of protein per ml. Final
volume, 1 ml. Incubation time, 120 minutes at 37”. The results are expressed aa
micromoles of creatine formed Der ml. of reaction mixture per 120 minutes.
Enzyme
ml.
0.025
0.06
0.1
0.1
0.1
0.1
0.1
1
Enzyme
2
Creatine
formed
ml.
0.15
0.16
0.15
0.15
0.025
0.05
0.1
0.0
0.8
0.975
0.975
0.0
0.32
0.66
0.85
Downloaded from http://www.jbc.org/ by guest on September 6, 2014
per cm; AYe 80
1.7
1.7
1.7
1.7
1.7
1.7
654
CREATINE
SYNTHESIS
Borsook and Dubnoff
(4) observed that addition of cyanide was inhibitory to creatine synthesis in liver homogenates.
Inasmuch as these
authors also found that, oxygen was an absolute requirement for creatine
synthesis, it was not clear whether cyanide inhibition was due to disruption
of the aerobic generation of energy-rich phosphate compounds or to interference with the transmethylation
reaction itself.
On reinvestigation
it
was found that, cyanide not only is not inhibitory but, in fact,, stimulated
the activity
of GA methylpherase.
In addition, other reducing compounds such as glutathione, cysteine, and BAL increased the activity of
the enzyme.
The activation
by -SH
and other reducing compounds
became more pronounced as the degree of purification
of the enzyme
Preliminary
treatment
of enzylne
I
GSH
I
* 0.001
M;
formed
+
3.04
4.83
0
+
4.93
None.................................
“ .. . . . . . . . . . . . . . . . . . . . . . . . .
. .. .
p-Chloromercuribenzoic
acid*.
.. .. .
“
“
(followed by GSH).
.
Creatine
45 minutes at room temperature.
increased.
Thus the activity of crude liver homogenates was not increased by the addition of -SH reagents; there was a moderate activation
of the initial
ammonium sulfate fraction and a very marked activation
of the alumina Gy eluates, or the ammonium sulfate fractions obtained
from them.
Further evidence for the dependence of GA methylpherase on
the presence of free -SH groups for activity was obtained by use of
p-chloromercuribenzoic acid. This reagent, at, a concentration of 1 X 1W
M
caused
complete
inhibition
of creatine
synthesis.
This
inhibition
could
be reversed quantitatively by subsequent addition of glutathione in sufficient, excess (Table V).
Ca* or Mg++ is not required in the reaction catalyzed by GA methylpherase;
sodium
fluoride,
folk acid, and Leuconostoc
citrovorum
factor
have no effect on the activity of the enzyme.
Evidence for Formation of Creatine-Studies of creatine synthesis have
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TABLE
V
Reversible Inactivation of G-uanidinoacetic Methylpherase by
p-Chlorometcwibenzoic
Acid
0.1 ml. of guanidinoacetate
methylpherase
enzyme, Ammonium Sulfate I containing 11.7 mg. of protein; Tris buffer, pH 7.4, 0.01 M; guanidinoacetic
acid, 0.003
M; S-adenosylmethionine,
0.004 M; glutathione,
0.005 M. Incubation time, 60 minutes at 37”. The results are expressed ae micromoles of creatine formed per ml. of
enzyme.
0.
L.
CANTON-I
AND
P.
J.
VIGNOS,
655
JR.
been severely handicapped by the lack of a specific micromethod for creatine determination.
Two methods are available for the determination of
creatine: one is based on the a-naphthol-diacetyl
reaction for creatine (13);
the other depends on the conversion of creatine to creatinine and determination of the latter by the Jaffe alkaline picrate reaction.
GA interferes in both determinations;
the degree of interference, however, is different.
Different also are the specificities of the two determinations.
It
is well known that the determination
of creatine, after conversion to
creatinine, by the Jaffe alkaline picrate reaction, is fraught with pitfalls.
In earlier studies (4, 5, 14, 15) one of the principal sources of difficulty
was the frequent occurrence in crude homogenates of cy-keto acids in gen-
Synthesis
VI
As Determined
by Two
Methods
0.3 ml. of GA methylpherase;
Ammonium
Sulfate
I containing
25.3 mg. of protein
in a final volume
of 1.0 ml.; AMe, 0.0018 M; GA, 0.0026 M; Tris buffer
(pH 7.4), 0.075
M; GSH, 0.001 M.
Incubation
time, 60 minutes
at 37”.
The results
are expressed
as micrograms
of creatine
formed
per ml. of reaction
mixture
per hour.
Creatine
Incubation
determination
time
Method
I’
Method
IIt
min.
0
60
Creatine
formed.
* Alkaline
picrate
t a-Naphthol-diacetyl
.
115
195
..
.
method
of Borsook
(11).
method
of Ennor
and
31.4
121
+80
Stocken
I
+89.6
(16).
era1 and of a-keto-y-methiolbutyric
acid in particular;
when treated with
alkaline picrate these cr-keto acids give rise to chromogenic products which
Treatment with
are indistinguishable
from those produced by creatinine.
Lloyd’s reagent, however,
appears to reduce greatly errors from these
sources (5).
Direct determination
of creatine by the cz-naphthol-diacetyl
reaction
is not particularly
suitable for routine use with this system because the
presence of -SH
groups interferes with the color development.
Such
interference
can be overcome, however,
by appropriate
treatment with
p-chloromercuribenzoic
acid, as suggested by Ennor and Stocken (16).
In view of the relative lack of specificity of the two methods it appeared
desirable to measure simultaneously
the formation of creatine by the two
methods.
Table VI shows the result of such an experiment.
The values
obtained for creatine synthesis as determined by the two methods agree
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TABLE
Creatine
656
CREATINE
SYNTHESIS
within 10 per cent. These results present strong support for identification
of creatine as one of the products of the GA methylpherase
reaction.
However, it was believed important to identify creatine more directly.
It is known that in the presence of creatinekinase,
ATP will phosphorylate
creatine to form creatine phosphate.
The substrate
specificity in this
case is very high, guanidinoacetate
and other guanidino compounds being
inactive in the system.
As is well known, phosphocreatine
is very labile
at acid pH and is completely hydrolyzed to orthophosphate
and creatine
during the course of the procedure employed for phosphorus determination
Additions
No additions......................................
Creatine,lfi;....................................
“
2
. . ..__.._._...............,_......._
Product of guanidinoacetate
methylpherase,
tion Mixture 2 (0.25 ml.)*. .
Reaction Mixture 1, controlt..
.
I
“Orthophosphate”
formed
0.55
11.00
18.90
Reac6.40
0.60
* Reaction Mixture 2 deproteinized
at end of incubation
and concentrated.
1
ml. contained 3.5 PM of creatine, as determined by the alkaline picrate method.
t Reaction Mixture 1 deproteinized
at zero time and concentrated to Bame volume
aa Reaction Mixture 2. Reaction Mixtures 1 and 2 are the same DB the complete syetern of Table II.
(17). Thus, if creatine were formed by the GA methylpherase reaction,
addition of a suitable aliquot of the incubated reaction mixture to creatine
kinase in the presence of ATP and Mg++ at pH 9.0 should result in the
formation of phosphocreatine which can be determined as “apparent orthophosphate.” Table VII describes the result of such an experiment from
which it is concluded that creatine is the product of the enzymatic methylation of GA.
Isolution of Reaction Product and Identijication As Creatinine Potassium
P&r&--The
purified enzyme (3 ml., 66 mg. of protein) was incubated with
AMe (190 PM), GA (380 PM), BAL (0.01 ml.), and glycylglycine buffer in a
final volume of 25 ml. for 4 hours at 37”. The reaction was terminated by
addition of 0.1 volume of 100 per cent trichloroacetic acid. After cen-
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TABLE
VII
of Creatine As Demonstrated Enzymatically
by Means
of Creatine Kinase
0.1 ml. of creatine kinase containing60
y of protein; ATP, 0.004 M; borate buffer,
pH 9.1,0.068 M; MgC12, 0.01 M. Additions a8 indicated.
Incubation time, 15 minutes at 37”. The results are expressed as micrograms of “orthophosphate”
formed
per ml. of reaction mixture.
Formation
G.
L.
CANTON1
AND
P.
J.
VIGNOS,
JR.
657
DISCUSSION
On the basis of the evidence presented it appears justified to conclude
that the enzymatic mechanisms involved in the biosynthesis of creatine
conform to the pattern revealed earlier in studies of the methylation of
nicotinamide.
The two systems differ, of course, in the specificity and
properties of the transmethylating
enzymes, but the mechanisms for activation of methionine and for the transfer of the methyl group are presumably the same in both cases. du Vigneaud et al. (2) have produced
conclusive evidence that in viva the methyl group of methionine is transferred as such to creatine. The exact mechanism underlying the migration
of the methyl group is not yet known with certainty; in view of recent findings on the structure of AMe, it would appear reasonable to postulate
that the methyl group might migrate as a positively charged methylcarbonium ion which could be transferred from the methyl donor either
directly to the substrate or first to the enzyme catalyst and then to the
substrate.
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trifugation, the supernatant solution was autoclaved in 0.05 N HCl for 30
minutes at 15 pounds pressure for conversion of the creatine present to
Next the creatinine was adsorbed on 1 gm. of Lloyd’s reagent
creatinine.
and eluted therefrom with 20 ml. of saturated BA(OH)z as described by
Bloch and Schoenheimer (18). In addition to creatinine the Lloyd eluate
contained an unidentified contaminant showing an ultraviolet absorption
with a maximum of 250 rnp. Prior to the crystallization
of creatine as
the potassium picrate double salt, it was deemed desirable to remove this
contaminant, since it also gave an insoluble derivative when treated with
picric acid. For this purpose the eluates were freed of barium, adjusted to
pH 7.8 with dilute phosphate buffer, and passed through a Dowex 50
(H+) column (15 X 45 mm.) which then was washed with 20 ml. of water.
For elution 0.1 N HCl in 0.1 N NaCl was used and the eluates were collected in 10 ml. lots. The fist three fractions were discarded and the
next nine were pooled. The combined eluates were adjusted to 0.1 per
cent with respect to both potassium picrate and picric acid and crystallization was allowed to proceed in the ice box. After repeated recrystallizations, approximately
15 mg. of the double salt were obtained.
An
aliquot was ground in Nujol and its infra-red absorption spectrum determined in a Perkin-Elmer
spectrophotometer.
The material exhibited a
spectrum practically identical to that given by an authentic sample of
creatine potassium picrate and clearly different from the corresponding
salt of guanidinoacetic
acid anhydride.
This provides further evidence
for the formation of creatine as the product of the enzymatic methylation
of guanidinoacetic acid.
658
CREATINE
SYNTHESIS
SUMMARY
1. The methylation of guanidinoacetic acid by S-adenosylmethionine
to
form creatine has been studied in partially purified preparations of pork
liver.
2. The enzyme, which is referred to as guanidinoacetate
methylpherase,
has been partially purified and some of its properties have been investigated. The partially purified enzyme requires reduced thiol groups for
optimal activity.
3. Creatine has been identified as a reaction product (a) by simultaneous
determinations
by two different methods, (b) enzymatically
by means of
creatine kinase, and (c) by conversion to creatinine and the isolation of
creatinine potassium picrate.
4 Hydrogen
ion formation
was measured
experimentally
by running
the reaction
in a Warburg
vessel and using
bicarbonate-CO2
buffers. It was clearly evident
that H+ formation
accompanied
creatine
synthesis,
but the exact stoichiometric
relationships
could not be worked
out because
of the limited
sensitivity
of the method
and the large amount
of protein
required.
6 E. Scarano
and G. L. Cantoni,
to be published.
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A comparison of Reactions 2 and 3 indicated that H+ formation* accompanies the methylation of GA, but not that of nicotinamide.
This is
related to the fact that in the methylation of GA a tertiary amine is formed,
whereas the methylation of nicotinamide results in the formation of a new
onium compound containing a methylpyridinium
bond.
Liener and Schultze (19), Stekol et al. (20), and others (21, 22) have
reported that vitamin B 12- or folic acid-deficient rats show decreased ability
to methylate, as indicated by lowered creatine and N1-methylnicotinamide
synthesis.
Since the r6le of vitamin Blz and of folic acid, or its derivative,
L. cilrovorum factor, in the biosynthesis
of methyl groups and in the synthesis of purines is well established, it would appear that these results
might be related to decreased synthesis of methyl groups or of adenine
nucleotides in the deficient animal.
This conclusion is supported by the
observation reported above that L. CdTOVOTUm
factor, folic acid, or vitamin
B12 had no effect on the enzymatic synthesis of creatine.
However,
the
possibility
that the enzyme guanidinoacetate
methylpherase
contains
tightly bound L. citrovwum
factor as its prosthetic
group has not been
ruled out.
It would be anticipated that the product of demethylation
of AMe
should be adenosylhomocysteine.
In fact, preliminary
results6 indicate
that a compound having chemical properties expected for ASR can be
detected by means of chromatographic
techniques following
the transmethylation
reaction.
Further
work aiming at the isolation and characterization of this demethylation
product is in progress in this laboratory.
G.
4. The mechanism
L.
CANTON1
underlying
AND
P.
J.
VIGNOS,
JR.
659
creatine biosynthesis has been discussed.
The authors are very grateful to Dr. H. Hirschmann and Mr. John
Corcoran for their help in the determination
of the infra-red spectra and
in the interpretation
of the results obtained by this technique.
It is a
pleasure also to acknowledge the technical assistance of Mr. Robert R.
Vaughn.
BIBLIOGRAPFIY
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