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Electronic Theses and Dissertations
1972
Studies on Reaction of Mimosine with Various Amines and Effects of Mimosine on Tyrosine Decarboxylation
Paul D. Ballata
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Recommended Citation Ballata, Paul D., "Studies on Reaction of Mimosine with Various Amines and Effects of Mimosine on Tyrosine Decarboxylation" (1972). Electronic Theses and Dissertations. 4625. https://openprairie.sdstate.edu/etd/4625
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OF MIMOSINE ON TYROSINE DECARBOXYLATION
BY
D. BALLATA PAUL
A thesis submitted in partial fullfillment of the requirements for the
degree Master of Science, Majo:r in _ __ Chemistry, South Dakota State University
1972
SOUTH D OTA STATE UN_l ERSITY LIB ARY STIJDIES ON REACJION OF MIMJSINE WITH VARIOUS AMINES AND EFFECTS OF MIMOSINE ON TYROSINE DECARBOXYLATION
This thesis is approved as a creditable and independent investigation by a cand idate for the degree, Master of Science, and is acceptable for meeting the thesis requirements for this degree. Acceptance of thi s thesis does not imply that the conclusions reached by the candidate are necessarily the conclusions of the major department .
Thesis Adviser Date
Head , Chemistry Department Date ..,. ;
DEDICATION
To she who walks in beauty
"Phyllis"
Acknowledgment s
The author wishes to express his appreciat ion to Dr . John Grove for his assistance with the conception, development , and culmination of this research and part icularly "for his patience during the writing of this thesis. The author al so wi shes to thank other members of the faculty for their helpful advice given throughout this research and
Mrs. Maris Knutson who typed this thesis. TABLE OF CONTENTS
I NTR.ODUCTI ON ••••••••••••••••••••••• • •••••••••••••••••••••••••••••••••1
L::�RATURE REVIEW
Mirnosine ••••••••••••••••••••••••••••••••••••••••••••••••••••••••3
Pyridoxal-5-phosphate ••••••••••••••••••••••• ••••••••••••••••••••7 EXPERIMENTAL
Apparatus and Materials ••••••••••••••••••••••••••••••••••••••••12
Isolation and Purification of Mimosine •••••••••••••••••••••••••12
Tyro sine Decarboxylase Studies •••••••••••••••••••••••••••••••••14
Effect of L-Canaline on Mimo sine-induced Decarboxylation •••••••15
Ultraviolet Studies ••••••••••••••••••• ••••••.••••••••••••••••••1 6
a ••••••••••••••• •••••••••• n-Butyl mine and Mimo sine Interaction � 17
Sephadex G-10 Chromatography ••••••••••••••••••••••••• ••••••••••17
Paper Chromatography ••••••••••••••••••••• ••••••••••••••••••••••18 RESULTS
Characterization of I solated Mimosine ••••••••••••••••••••••••••20
Tyrosine Decarboxylase Results •••••••••••••••••••• •••••••••••••20
Effect of L-Canaline on Mimosine Catalyzed Reaction••••• •••••••29
Ultraviolet Studies ••••••• ••••••••••••••••••• • •••29
Sepha_ dex G-10 Chromatography •••••••••••••••••••••••••••••••••••34
Paper Chromatography ••••••••••••••••••••••••••••••••••••••••••• 40
DISCUSSION •••••••••••••••••••••••••••••••••••• •••••••••••••••••• ••••4 7
•••••••••••••••••• ...... •••• 6 SUMM.ARY ...... � ...... 5
•••••••••••••••••.•••••••• ...... • •• APPENDIX ...... 58
REI=ERENCES •••••••••••• ••••••••••••••••••••••••••••••••••••••••••••••6 0 LIST OF FIGURES
Figure Page . 1. 21-22 Infrared Spectrum of Purified Mimosine ••••••••••••••••••• 2. Reciprocal Plot of Tyrosine Decarboxylat ion Reaction Rates with Variable Mimosine Concentrat ion and Constant Tyrosine Concentrat ion...... 27
3. Reciprocal Plot of Tyrosine Decarboxylat ion Reaction Rates with Variable Tyrosine Concentration and Constant Mimosine Concentration...... 28 4. J0-31 Ultraviolet Spectrum of Iso lated Mimosine •• •••••••••••••• 5. Ultraviolet Spectra of the Reaction of Mimosine and 32-33 L -Valine at Varying L -Val ine Concentrat ion •••••••••••••• • 6. Ultraviolet Spectra for the Reaction Between n-Butylamine 35-36 and Mimosine•• ••••••••••••••••• •• •••••• •••••••••••• •••••• 7. Infrared Spectrum of Residue from Mixture of Mimo sine and n-Butylamine in Benzene...... 37 8. 38-39 Ultraviolet Spectrum of Mimosine�Mimosine Reaction ••••••• 9. Sephadex G-10 Column Fractions...... 41
10. Radiochromatogram of Timed Reaction Mixture of 14C-Tyrosine Mimosine, and Enzyme...... 44
11. Paper C0romatogram of Reaction Mixture and Fraction 49 from Sephadex G-10 Column...... 46 .r -...
LIST OF TABLES
Table Page
I. Conditions and Representat ion Reaction Rates for Tyrosine Decarboxylase Manometric Assays ••••••• •••••••••••2 3
II. Tyros ine Decarboxylation Reaction Rates with Varvinq Mimosine Concentrat ions + Constant Tyros ine Concentration •••••••••••••••••••••••••••••••••••••••••••••25
III. Tyrosine Decarboxylat ion Reaction Rates with Varying
Tyrosine Concentrations + Constant Mimo sine Concentrat ion ••••••••••••••••••••••••••••••• ••••••••••••••26
IV. Paper Chromatogram Rf Values fo� Known Compound s Detected with Diazotized sulfanilic acid - NH40H Spray •••••••••••••4 2
V. Paper Chromatogram Rf 'Values for Known and Unknown Components of Tyrosine Decarboxylase Reaction Mixture •••••43 1
IN1RODUCTION
As early as 1897 the toxic effects of mimosine were observed and animals ingesting mimo sine showed symptoms of hair loss, infertility, and retardation of growth. 1 Mimosine occurs naturally in leguminous plants ind igenous throughout the tropics of the world. Since the forage of these plants ha s a protein content as high as al falfa and is reli shed by all kinds of livestock, 2 the mechani sm of the toxicity of mimo sine should be explained .
Although the effects of mimosine have been known for a long time, the reason or mechanism fo� these effects ha s not been explained. Mu ch work ha s been done on the po ssible interference of mimosine with the incorporation of certain amino acids into proteins.
How mimosine may cause its toxic effects by the inhibition of metal-
Gontaining enzymes or by complexing with pyridoxal-5-phosphate has also been studied. However, this work has not yielded any conclusive mechanism for mimosine's toxicity. Because of varied result s, the observation that mimosine-induced growth inhibition could be reduced or completely eliminated by supplying animals with additional amino acids has not clarified the mechanism for the toxic. effects of mimosine. Therefore , this research wa s undertaken to help clarify the mechanism for the adverse effects of mimo sine.
It wa s noted that mimo sine structurally ha s many of the same functional groups as pyridoxal , including an ortho hydroxyl group and an oxygen of a carbonyl group being in conjugation with the ring nitrogen. Because of thi s similarity to pyridoxal, experiments were ""' --- 2 done to attempt to show that this resemblance is functionally important and may be the main cause for the toxic effects of mimosine.
0 I I H ()° 0 I . -{;H-C-OHJI CH 2 NHr 2
Mimosine Pyridoxal 3
LITERATURE REVIEW
Mimosine or 3-hydro xy-4-ox�-1(4H)-pyridine alanine
Loss of hair in animals following ingestion of seeds and foliage
of Leucaena glauca was first reported in. 1897. 1 Inge stion of mimosine
by sheep not only causes the loss of wool but may eventual ly cause the
animal to die. 3 Mirnosine ha s been reported to completely inhibit the 4 growth of coli. The cessation of the estrous cycle and eventual E. complete infertility of female rats has also been attributed to
mimosine ingestion.5 Sudden loss of ha ir in native women has been 6 ascribed to consumption of Leucaena qlauca seeds. Two papers have 7 described the inhibition of growth of hair by mimosine and other
effects caused by mimo sine in several animals.8
In 1950 Malmquist showed that Leucaena glauca did in fact contain
mimosine as an unbound amino acid. 9 The structure of mimosine was 10 first proposed by Wibaut and proved by Adams and Johnson. 11 Organi c
synthesis of mimosine was reported by Spenser and Notation in 1962
from J-benzyloxy-4-pyrone and -amino-�-tosylaminopropionic acid. 12 .B Much work on the bio synthesis of mimosine has been done during . the past ten years. Hylin in 1963 showed that the .administration of 14 2- c) -DL-lysine to Leucaena glauca results in the incorporation of � radioactivity into the pyridone ring of mimosine. 13 Notation and 14 Spenser in 1964 also showed that C-labelled aspart ic acid was
incorporated into the pyridone nuc leus of mimosine . 14 In 1965 Tiwari
and Spenser reported that succinic acid was incorporated into the
nucleus; while glycerol and glyceric acid contributed label to the 4
side chain of mimosine. In it wa s determined that ser ine 15 1967 serves as a precursor of the alanyl side-chain of mimosine and that
o<.-aminoadipi c acid is incorporated into the pyridine nucleus. 16
Just recently it has been .shown that an extract of Leucaena
glaucaseedlings can_ catalyze an enzymatic synthesis of mimo sine from
3,4-dihydroxypyridine and o(-acetylserine. They also reported that
neither serine nor , -diaminopropionic acid served as direct o( /) precursors in the in vitro enzymatic reaction. U se of a boi led
ex tra c t y1e . ld e d no m1mo. s1ne. . 17 It was observed that feeding ferrous sulfate to animals at the
same time that mimo sine wa s being ingested reduced the toxicity of
mimosine. It wa s suggested by Mat sumoto that the effect of ferrous
sulfate in reducing the toxicity of mimosine wa s due to the poor
H� absorpt ion of the ferrous complex o f mimo. sine.
Becau se of this complexing of mimosine with the ferrous ion, the to�icity of mimosine was also proposed to be caused by mimosine's
complexing with other metal ions required by various enzyme systems.
However, Lin et al. suggested that the toxicity of mimosine in vivo has no neces sary relationship to the chelating ability of mimosine's pheno lic hydroxyl group. 19
The inhibitory effect of mimo sine on many pyridoxal-requiring enzyme sy stems has been attributed to the reaction of mimo sine with pyridoxal-5-phosphate. The following reaction of mimosine and 5 pyridoxal-5-phosphate wa s also
+
Mimosine
However, it has been reported that the mimosine-pyridoxal -5-phosphate 20 interaction is less biologically significant in vivo� Lin et al. also reported that the pyridoxal pho sphate content in the tissue of mimo sine-treated rats was not less than that of control animals. "0
Hylin and Lichton po inted out that the role of vitamin B6 in the biosynthesis of many amino acid s is well documented and suggested . the possibility that an antagonism of vitamin B6 may account for the 5 observed effects of mimosine.
Because of the restricted growth of hair and wool caused by mimo sine, it has been postulated that mimosine may affect -some vital common step involved in the synthesis of such fibrous proteins and perhaps protein synthesis in general. Since mimo sine structurally resembles tyro sine more closely than any other protein constituent, it has been suggested that it acts as a tyrosine analog�e. 21
TI-le toxic effect of mimo sine in rats can be reversed if supple- ments of tyro sine are added to the diet. Phenylalanine can also 6
22 partially reverse mimo sine 's effect . It ha s been previously
sugge sted that mimo sine may interfere with the metabolism of tyrosine.7 ·Smith and Fowden reported that mimosine did not restrict the incorporation of phenylal nine into protein nor did mimosine replace a 23 phenylalanine in protein mo lecules. The incorporation of 1'+c- leucine into newly synthesized protein wa s also not affe cted by mimosine.24
Much work has been done on mimo sine's inhibition of enzyme systems. It has been reported that mimosin- e caused inhibition of pheno l oxidase, tyro sinase, mammalian and plant phenolase, catala se, 6 and alkaline phosphatase systems.25'2
It has been established that mimosine stimulates ATP-PPi exchange
the pre sence of phenylalanyl-s-RNA synthetase from mung bean plant.23 in Lin et al. have reported mimo sine's effect on three enzyme 20 sy stems. Glutami c decarboxylase wa s not signifi cantly inhibited by mimo sine or L-glutamic acid but was inhibited by DL-phenylalanine.
It wa s also noted that mimo sine cancels the inhibition caused by phenylalanine. The activity of L-dihydroxy-phenylalanine DOPA) ( decarboxyla se apoenzyme wa s increased in the pre sence of mimosine but wa s decreased if pyridoxal pho sphate was added. DL-phenylalanine and _ �:-9lutamic acid inhibited DOPA de carboxylase. Thi s inhibition was not reversed by addition of mimosine to the diet and was in fact increased.
With gl�tamic-oxaloacetic-transaminase mimosine had no significant effect. Phenylalanine caused a marked induct ion of the enzyme. Thi s ,,. --
7 induction.could. be comple:te ly abo lished by the administration of
mlmosine. L-Glutamic acid inhibited the enzyme a�d this .. inhibit:j.o�
· was +eversed by mimo sine. The me chanism for the reversal effects
ca\.lsed by mimosine wa s not described.
Lin, Lin, and Ling whi le studying the effects of mimo sine on L -
DOPA decarboxylase found that at low concentrat ions of mimosine, 1 .7x
10-5 M, an i'ncreased rate of decarboxylat ion of L-OOPA occurred.27
Thi s fact wa s not explained. It should be poi nted out that at high
concentrations, around l.Ox10-3 M, mimosine essential ly complexes
with all avai lable pyridoxal thus cau sing complete inhibition of _ pyridoxal requiring enzyme s.27 Therefore, the activation of L-DOPA
decarboxylase by mimosine at low concentrations is hard to explain.
Pyridoxal - 5 - Pho sphate 8 The activity of vitamin B6 was first defined in 1934 by Gyorgy. 2
Then in 1938 five research groups iso lated crystalline salts of B6 , 29 Gyorgyst being one of these. The structure of pyridoxol was independ-
ently established by two groups in 1939. 30'31 A new pyridoxol-like
substance with greater growth-promoting activity wa s proposed by Snell
et al. in 1942.n The synthesis of the biologicall:y active aldehyde . 33 form of B6 was reported in 1944.
When tyrosine decarboxylase wa s first iso lated from � f aecalis g,
its coenzyme portion was characterized as a pyridoxal derivative . 34
It was soon shown that for a(-amino acid decarboxylases in general the
coenzyme wa s this pyridoxal derivat ive. Thi s functf�nal pyridoxal
derivative wa s designated pyridoxal pho sphate.35 It wa s reported by
Snell in 1945 that pyridoxal pho sphate wa s the coenzyme for trans- 8
6 aminases .3 o<., -Elimination reactions were also shown to be
catalyzed by pyridoxale phosphate.37,3g
The general mechanism by which pyridoxal pho sphate catalyzes the . ' 9 reactions of o<.-amino acids was proposed by Metzle-r et in 3 aL i954 . Mechanism
base intermediate Schiff
+
amine and pyridoxal. -+ It wa s pointed out that the formyl group of pyridoxal pho sphate
functions in the formation of a Schiff base with amino acids. This
Schiff base intermediate is stabilized by chelation with a metal ion which promotes planarity in the conjugated system. concerted A electrophilic action by the base ring-nitrogen atom of the pyridoxal moiety and the chelated metal ion is transmitted through the conjugated system, ·thus. potentiating the displacement of an electron pair from the o(-carbon atom of the amino acid . 40 The displacement of the O' --
9
electron pa r C?n occur between the H atom and the o<. carbon atom a , � - ( ) between the ci... carbon and the carboxyl group b , a11:d between the - ( ) alkyl group and the � carbon atom c . - ( )
H c I a b amino acid group R - C - COOH
NII C-H f ormyl group f j HOH C OH 2 pyridoxal mo iety ..-: . CH 3 QN This displacement of anJ ele ctron pair or rupture of any of the a, b, or c bond s has been report�d to be accompanied by an increase of
the resonance energy of the Schiff base intermediate. Thi s increase 4 of the resonance energy is in the order of 8 Kcal mole. 1 / Spectrophotometric evidence for the Schiff base formation between
pyridoxal phosphate and glycine was first reported by Blakely in 2 1955.4 Metzler demonstrated an equilibrium between pyridoxal and amino acids and the ir imines using spectrophotometric methods in
1957.43 3 Metzler et al. 9 propo sed that the structural features of the pyridoxal mo lecule essential for catalysis of non-enzymatic reactions are the aldehyde group ortho to an hydroxyl group and the aldehyde group being conjugated with a heterocyclic ring nitrogen or an electr0n withdrawing group. Dowhan and Snell in 197044 clearly showed that the 2-methyl group and the unsubstituted 6-po sition of pyridoxal phosphate are not essential for its activity . The 2-methyl group can be 10 replaced by a H atom without any inactivation at all. Thi s fact,
the 2-methyl not bei ng essential, has now been reported for many 4 enzyme systems requiring B6 coenzyme. 5 6 With pyridoxamine - pyruvate transaminase4 and serine dehydra�
tase, 44 both the phenolic hydroxyl group at C-3 and a free pyridine
nitrogen are required for the binding of pyridoxal at the active site
of the enzyme. Other evidence suggests that these groups may partici
pate in catalytic events with the en�yme-substrate complex.
It has been propo sed that pyridoxa1 phosphate may bind to enzymes
by the following methods:47 1) the pyridoxal phosphate may form a
covalent aldimine bond with the -amino group of a lysyl residue on ¢ the enzyme and by ionic bonding between the pho sphate group and the
enzyme, 2) the binding of the pyridine nitrogen atom to a proton
donating group of the enzyme, and 3) the interaction between the
enzyme and both the pheno lic oxygen and the heterocyclic nitrogen.
Snell also suggested that the binding of pyridoxal pho sphate to the
enzyme may occur by hydrogen bonding and physical bonding (electron
cloud interaction) through the interaction of the aromatic ring and
the enzyme. 45
It has also been well documented that rather extensive conforma
tional changes accompany resolution of several pyrido xal pho sphate
enzymes. These structural changes affect not only the catalytic effectiveness of the enzyme, but also the binding capacity, the rate
of binding, and the capacity to produce.the essential conformational
changes.45
L-Canaline strongly inhibits the activity of nearly all pyridoxal- v --..
11
dependent enzymes. It has been reported that canaline probably inhibit s pyridoxal pho sphate - containing enzymes by its nonenzy matic , irreversible, and stoichiometric binding with pyridoxal phosphate. This inhibition is probably caused by the oxime or Schiff 4g base formation of L-canaline with pyridoxal pho sphate. ..,. -.
12
EXPERIMENTAL
Apparatus
The following apparatus were used during this research:
Fisher - Johns melting point apparatus
Perkin - Elmer models 700 and 521 IR Spectrophotometers Finnigan Mass Spectrometer model 3CX)O, quadrupole type
Sargent model LS pH meter
Beckman model DK-2A Spectrophotometer Circular Warburg apparatus
ISCO fraction collector model 272 with an ISCO volumeter model 400
Packard Tri-Carb liquid scintillation counter model 3375
Packard Radiochromatogram Scanner model 385
Eberbach water - bath shaker Materials
Tyrosine decarboxylase apoenzyme wa s obtained from Worthington
Biochemical Corp.; L-mimosine was obtained from Calbiochem or iso -
lated; Sephadex from Pharmacia Fine Chemicals Inc .; L tyrosine G-10 - from Fisher Scientific Co.; .Uniformly -labeled 14C -tyro sine, 2. 26 pC/ug, .from New England Nuclear. Pyridoxal-5-phosphate, tyramine , dopamine, _ p-hydroxylphenylpyruvic acid, and L-canaline were obtained from Sigma
Chemical Co.
Isolation and Purificat ion of Mimosine Seeds of Leucaena glauca ( Koa Haole) were ground to a fine powder coats were separated from the in a No . 2 W iley Mill. The hard seed mixture of of seed me al by repeated decantation of a stirred 50 g. ,, -
13 ground seeds· and 1250 ml of distilled water. It should be noted that all the seed coats must be removed, otherwise pure mimo sine cannot of be obtained. Cation-exchanger Rexyn 101 H , laJ ml of settled resin, ( ) was added to the mixture of water and seed meal and was stirred for 24
hours. The meal wa s then decanted leaving only the re sin which was
transferred to a glass column (2 x 40 cm), and the mimo sine was
eluted by the fol lowing method.
The column was washed with one liter of distilled water fol lowed by 4CXJ ml of 50% ethano l in order to remove the organic contaminant s.
The ethanol was eluted with water and mimosine was displaced with
2 N ammonium hydroxide ml) , the eluate being collected after the (600 ammonium hydr.oxide front was half way down the resin bed. The eluate ° was concentrated to a thick syrup at 40 c in a rotary evaporator • .
The syrup was dissolved in ml of water and the solution adjusted 20 to pH 4.5 with N HC l. The suspension was stored overnight at 4°c 6 and the crystalline mimosine was collected by filtration, washed with
cold water, and dried.3
The crude mimo sine was made into a hydrochloride salt by adding two equivalents of HCl for each equivalent of mimosine and by evapora ting the mixture to dryness under a vacuum. The salt was recrystal lized from ethano l using at least twelve equiva lents of ethanol to each equivalent of salt. The recrystallization mixture was allowed to stand overnight in the dark at 4°c. If the recrystallization was done in light, the product turned purple. The product was then dried by suction and put on a Dowex 50-H+ ( 8X) column x 40 cm as a sol id. (2 )
272109 LIBRARY ATE UNIVERSITY SOUTH DAKOTA ST 14 Water was applied to the column until all of the sample wa s dis solved.
The column was then washed with additional water. When putting the product on the column the water mu st be applied without int errupting its flow until at least 500 ml of distilled water have been collected; otherwise the purity of the product is decreased . The column was then wa shed with 400 ml of ·50% ethano l and the mimosine wa s eluted as described above .
Tyrosine Decarboxylase Studies
The effect of mimosine on the rate of decarboxylation of L-tyro sine catalyzed by tyrosine decarboxylase ( E. C. 4.1.1.25) was determined by fol lowing the liberation of C0 2 as mea sured with a Warburg apparatus. All reaction systems were buffered to pH 5.5 with a 0.5 M sodium acetate buffer. The reaction volumes were held constant at 3.0 ml. All the systems were pre-incubated in the Warburg for 15 minutes before the reactions were started. The Warburg was always maintained at 37°c. The reactions were started by dumping the content s of th side arm usually tyrosine or a mimosine-tyrosine _ � ( mixture into the main compartment of the flask . all systems the ) In main compartment of the Warburg flask contained -one ml of buffered enzyme prepared by adding mg of lyophilized enzyme to 4 ml of 0.5 M ( 1 acetate buffer, pH 5.5) and 0.5 ml of a pyridoxal-5-phosphate solut ion -6 such that the final pyridoxal concentration was 1.67 x 10 M. Buff er and distilled water were added in various amounts in-order to maintain a constant vo lume of 3.0 ml. Because of the complexing of mimosine witp pyridoxal care must be taken not to combine the se two reagents ... - --
15
prior to the start of the experiment. ·The amount of evolved C02 was determined at 10 , . and· 35 minutes. 5, 15, 20, ·25 The rate of decarboxylation of 1 4C-tyrosine at a very dilute
concentrat ion, x 10-7 M 3$),000 dpm was determined by trapping 4.0 ( ) the 14c02 on a wick of Hydroxide of Hyamine . The reactions were
carried out in 50 ml Erl enmeyer flasks fitted with a center well.
For all of the sy stems studied in thi s series of experiment s one ml
of diluted enzyme ( prepared by adding mg of enzyme to 8 ml of M 1 0.5 acetate buffer, pH one ml of buffer , ml of water, and 0.5 ml 5.5), 0.25 1 6 6 of x io- M mi mosine or 0. 5 ml of 1 x 10- M pyridoxa l were added to the Erlenmeyer flasks. Wicks of Wh itman No . 1 paper were saturated with Hydroxide of Hyamine 10-X and placed in glass cups. These glass
cups were then inserted into the center wel ls. The flasks were sealed
with a sleeve -type rubber stopper and -pre•incubated at 37°C in a water- bath shaker for 15 minutes. The reactions were initiated by injecting ml of 14C-tyrosine ( 3$),000 dpm) into the reaction mixtures. The 0.25 reactions were stopped ·at various times injecting one ml of N HCl by 6 into the fl�sk. After �tanding for a· ha lf-hour the glass cups cont ai�
ing the trapped 1 4co2 were removed and placed in a scintillation vial containing ml of ethanol-tolene-POp-POPOP scintillator ( 1.5 L: 10 L: g: g�). The amount of· 14co wa s determined by a 2.25 1.5 0.75 2
· liquid scintillation counter.
The Effect of L-Canaline on Mimosine-induced Decarboxylation - - - The rate of decarboxylation of a mimo sine + L-canaline reaction 4 was determined in the same manner as described fqr dilute 1 C-tyrosine. 16
0.5 1 0 0.1 The reaction consisted of ml of xl -6 M mimosine, ml of 0.1 M L-canaline, 0.15 ml of water, one ml of buffer, one ml of
enzyme ( prepared by adding 1 mg of enzyme to 8 ml of buffer), and
6.25 ml of 1.22 x 10-6 M (190,000 dpm) 14C-tyrosine. The mimosine ,
L-canaline, water, and buffer were incubated for one hour in the 4 Erlenmeyer flask before the enzyme and 1 C-tyrosine were added. The
reaction was stopped after two hour s with one ml of 6 N HCl.
Ultraviolet Study 2i. Mimo sine - L-Valine- Interaction The Beckman DK-2A spectrophotometer was standardized using 49 potassium dichromate in 0.01 N sulfuric acid. The reaction solu-
tions were adjusted to the desired pH with dilute sodium hydroxide or dilute hydrochloric acid.
The reaction between mimosine at a constant concentration of ·
10-5 M and L-valine at varying concentrations (G.005 M, 0.0 1 M, 5 x 0.0125 M, and 0.025 M) was studied. These reactions were carried out at the three pH's (5. 5, 7.0 and 10 .5). The samples were prepared by
50 1 50 adding ml of x io-4 M mimo sine to ml of the desired L-valine concentration and allowing them to st and at room temperature for four hours before the absorbance was read. The absorbances were recorded against a reference solution of mimo sine wh ich had the same concentra
(5 tion x io-5 M) as the sample. The absorbances of solutions of mimosine and L-valine were also recorded separately against water as
the reference cell. In addition the absorbance of an old (5 days) mimosine solution was recorded against a freshly-made mimosine
solution. 17
n-Butyl amine and Mimosine Interact ion
(J.69 A large molar excess of n-butylamine g) was added to 0.5 g of mimo:_s.ine in _ 100 ml of benzene and the mixture was refluxed for two
hours. The formation of water was ob served during the refluxing. The reaction mixture was then filtered to remove the unreacted mimosine
and was evaporated to dryness in vacuo at l00°C. The dried residue was dissolved in 50 ml of water and an ultraviolet _spectrum was · obtained. The solution was then evaporated to dryness and a KBr
pellet made from the residue. Sephadex G-10 Chromatography
The preparation of the column was as described by Eaker and 50 Porath. A sample of 5).19 g of Sephadex G-10 was suspended in 400ml of 50% (by vo lume) of glacial acetic acid in a �O ml suction flask and degassed thoroughly with an aspirator. The gel was allowed to settle and the liquid decanted. The gel wa s then resuspended in
4CXJ ml of 50% glacial acetic acid , degas sed , and decanted . This process wa s repeated three times. The gel was washed once with 400ml of distil led water and finally equil ibrated in 400 ml of O. 2M acetic acid containing 1% NaCl. The gel wa s allowed to stand overnight to insure complete swelling. The gel slurry was then packed into a tle for three days. After 1 x 150 cm glass column and allowed to set settling th9 column wa s washed with distilled water and six volumes of gassed distilled ter. 1 M pyridine followed by three volumes of de wa The vo id volume was determined by blue dextran. The column was standardized with respect to tyrosine and mirnosine 18
by applying a ml sample consisting of 4.5 ml of M mimosine and 5 0.05 .· . ml dpm) of 1 4C-tyros ine to the column and eluting the o 5 (100�000 sample with dega ssed distilled water. One ml fractions were collected
by -a fract ion collector. Starting with the vo id volume ( fraction
number all odd-numbered fractions were tested for 14C using the 41)', liquid scintillation counter. The even-numbered fractions were tested
with ninhydrin for the location of the mimosine fraction. Reaction mixtures of one ml of enzyme mg of enzyme to ml of (1 4 buffer), one ml of buffer , ml of M of mimosine , and of 0.5 0.05 0.5 ml· 1 4c-tyros ine were applied to the Sephadex column after (100,000dpm) filtering through filter paper. The pyridoxal pho sphate and the
enzyme had been added or eliminated to give a desired reaction. The reaction times were also varied. After the reaction mixture was applied to the Sephadex column, the different column fractions were
analyzed by . paper chromatography.
Paper Chromatography One-dimensional paper chromatograms were deve loped by the ascend - . ing No. paper was used with a n-butanol: glacial technique. Whitman 1 acetic acid: water v/v/v) solvent system. The dry developed (60:15:25, 14 chromatograms were fir st analyzed for C by th� radiochromatagram scan-
�er then sprayed with a ninhydrin solut ion to detect the unlabeled.amino id - spray produced acids. Because �he diazotized sulfanil�c ac NH40H characteristic colors with the reaction products and mimosine, it ·was
used as the indicator in l ater chromatograms instead of ninhydrin. The developed ch romatograms were first sprayed with diazotized sulf - 19 anilic acid ,_ then dryed and over-sprayed with dilute ammonium hydrox ide to p�oduce the characteristic colors.51 The diazotized sulfanilic
acid spray wa s made just prior to spraying by mixing 8.34 ml of A-1 0.5 ml of at consisted of 0.3 g of sulfanilic acid, to A-2 o0c. A-1 22.0 ml of concentrated HCl, and ml of water (0 .3% sulfanilic acid 78 in HC l) . consisted of 0.5 g of sodium nitrite and 10.0 ml of 8% A-2 water (5% NaN02 ). Rf values of standard s of mimosine, tyrosine, 14C-tyrosine , dopamine, p-hydroxylphenylpyruvic acid , and tyramin� ,,.,. .... were determined. Standards were applied to the same spot as the reaction mixtures for identification purposes. .., ·-
RESULTS
Characterizat ion of Isolated Mimosine
Starting with ground seeds of Leucaena glauca, yields of 3-5% of purified mimosine were obtained. The me lting po int for the puri - fied mimosine obtained was 223-224°c (decomp.). mixed melting A point with a known sample of L-mimosine was 221-224°c. The infrared absorption peaks for the isolated mimo·sine using a KBr pellet were
3230 cm- 1 ( OH), 2820 - 1 (NH) , 1640 - 1 1580 · - 1 aromatic cm cm = O), cm � (C - - (COO ), 1530 cm- 1 (NH 3+ ), 1350 cm- 1 ( O�··H-0-), and 1230 cm- 1 (Fig. 1). The spectrum corresponded with that reported 0) ( �:;c= 2 by Spenser and Notation. 1 By paper chromatography the isolated . 0.22 0.02 mimo sine showed a single spot with an Rf of :!:. when treated with ninhydrin. mass spectrum indicated a mo lecular ion of 198. A Tyrosine Decarboxylase Results
The various reaction mixtures ( Table 1) were tested for m2 production by the Warburg method. The react ion rate during the first 15 minute period subsequent to a pre -incubat ion of mimosine with tyrosine in the side arm was · greatly increased (3.468 pl C02/min. ) compared to a contro l
(1.692 plC02/min. ). The extent of the rate increase depended on the concentration of mimosine and the length of time that mimosine and tyro sine \":ere pre -incubated. Because it was once postulated that mi mosine facilitated the conversion of tyrosine into its keto acid form (p-hydroxylphenyl- pyruvic acid) which then would be more easily decarboxylated, a REMARKS Mimosine ORIGIN Isula:ted mjma'3jce h:cm PERKIN-ELMER Leucaena glauca MODEL 700 0 SPECTRUM NO.
OH PURITY Homog�nQui;z JI ga12er on cbrQma- SAMPLE Purified 1 MimQsine togro.pby pellet 0 PHASE KBr N 0 CONCENTRATION mg to KBr CHiI H-COHfl 1 l 'JJ mg THICKNESS SAMPLE iNH2 2
DATE I 26 AQ�il 12'.Zl OPERATOR P. D. B.
FREQUENCY (CM·1> .tOCO 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650
100' I 0,0 · I I I • l I I ·-··- ... : . .... ' I - I I ' . I I I I I 1 - 1 t i i .1 , . -· �-·- -1 -r1 ... l . L :-.J H· . .L. H , .. ).. ·- t �·- l -- . 1 . �I I rtI 11 L I l 1 ! ! I ! I I , ' I ! 1 I I . ' Ii I : I ! I I !I . I i I I � I i H 90 7 I - -- I I I . I I - . i I I I ' I - .... I .... I - I ! - 1 1 ... - . - .!- ·t . , _ I - - _ .,_ _ ''.L . . . I • I -1- I _,.... -t . ·- . � : ; i . . ·- --- r ' -1I _!. ·-i-+- , i I - I I I I : I ·: - ' I I I :. -l;H-I i I -HIJ � .. 80 I + h+ 4 F.... I : I I I I � I I - : : I · . • · • · • ltt' • -· I I I , i 11 I i' I i.! i Ir: ; l 1 l I 1 ·-- 11 'I I I . I I ' ! · I I I . I I 1 �- I 1 I 1-' I 7 - . . I . 0 , . I I ,, . , . I l - - - .. , .., I : l - , .... - ' • -4, . � : ' __ ! • o! i - - ·r � , I ' i ' ! I f-1- I T . ... i_ , fI r , : 't" '\r 'l.1._ o.1 I � -- L -- + i --� Zi'I r I \ ' I 11 I >.+t r1 • �} :- - --l· .... . 11 : - 60 -4- I l- -� • . -�. I I · t �!- . w 111 .!,_ : , , , : , , . , , , I , , f+" 1 nd l, i : r ; . 1 , •. I -t:..-rr- �_f • . - . , I T � 11 i I u , . 1 I j I : -t ti I . • I : I -- ) I ! I I j�v l 50 . ' ' I ! Ii . I I I � ,_1_ I I ' I I ' �I I o.J . I I I ! I l n i' -r-l------� • " · \I - · +l · ..... -�,_, ! ! : I I ! '. I ' . · I /: I l I : Li t: 1 : - 1 �' 1· -r · 1 i ·- i- ; · , ,_ . .. - ·.... - , . -i ·- - Iii J - �- ·1 r t, fi 1-.1 -1 --h1 rrt- . ' i --+-+- JUi I I I � 4 + I � I I I I i .J 1 I ' T I w· I I _ ! I I I 0 I ! ·* ,.. 0.¥ -:- . - . . � _ _ -:- � ! I . � 1 I +-. I -�f- I �- I I i .1 I I I 1 .1 ; � � "',J I I ' I 1 . UTI , I 1 1 � ' � -·, I I I t' I 0.¥ � 30 + .'1,� I �- I I I� 1- I ' � � , . r1 11 1 � ! rrI i- i , • �+ LI+�-,., f [ t·4t1- 1 ! I : I I ! ' ; - . I \..-., ! I I : I 1 I i I . i I : I ! ; I fi, I i ! I i ! .. - 11 J.t ! l . ... fl' rt ; T • ti I I I v I I TI : lj·, ·nrt r·;I . · i i · + lTTI I l I I -r--f-t-·-' 1--, I I I . I I I . ' L ' I ' .I ' I . H I I I i Vi I rt- 1�1I 20 I I 1 I . I I rrl . I .--L.I I� I IT ! I ! I, .. o., I I - . - -I J - I - ' I i1 . -·- I �._,, ; I I I I �I I . I I � 1 1 I I . 0.1 ·r· l .. t· . . L � . i. ·� � -j· I- -� � 1 I- i ; 1- ; �. . l �. I : r T . I - , ' -·r1- l r I • r' - I °'' I I -- -+I I i I ·. I ' ·--+ : � j I I I ! :� I , , I ! - 10 ' I t .�... l _! I ' E:. 1.0 I i I . I I . . I I l : ! ; I {: i I . . Vji I : '• I I • . + ! II : I' ;' : I ' 1' f ; L . ll - I I I : . I I ' 1I 1 • i I lt1· l l\J ' ; I '! I 11I I I I 1 I.i l I . I. I I: j I,,·-j I I I I I lt- 2.• 0 I l - i · r 17I I I I I I 11 n ' l\J ' 1 • • ;l.r 3.'o .J!.1 , "·" . .,.'.r J MICitoNi I 16 1a ·� 'o 1 1·111,'� 1 ttt' 11 14-1
� :�,, . Table Cond itions and Representative Reaction Rates for Tyrosine Decarboxylase Manometric Assays. I.
Ma in compartment Side arm Conditions Rate of Reaction
Enzyme + . Pyridoxal phosphate Tyrosine Designated as a standard reaction lC02 min. 1.692 )l /
Enzyme very litt le
+ or Mimosine Tyrosine For a 45 minute reaction time no reaction
Enzyme
+ Mimosine + MgC12 Tyrosine no reaction
Mimosine + Enzyme Mimosine and pyridoxal pre decreased rate incubated together for hour + by Pyridoxal phosphate Tyrosine 1 20-40%
Enzyme + Pyridoxal phosphate Mimosine no reaction
Enzyme Mimosine Mimosine and tyrosine pre incubated for hour. 3 4 8 lC02 min. + + 1 . 6 p / Pyridoxal pho sphate Tyrosine
l\) 'vJ f . ....
24
Warburg react ion was tried with p-hydroxylphenylpyruvic acid as the substrate. However, no decarboxylation took place. Therefore, mimosine does not convert tyrosine into its keto acid form.
At this po int a kinetic study was attem ted to define the inter- p relationship between tyrosine and rnimosine and to examine the reason for the increased rate. Two systems were employed in this study: the first holding the tyro sine concentration constant and varying the mimo sine concentration; the second holding the rnimo sine concentration constant and varying the tyro sine concentration. The resulting data were used to prepare Lineweaver-Burk reciprocal plots ( Tables II and III; Fig.2 and 3. The l V for both graphs was 0. 267 or V was 3.75 / pl C0 min . 2/ A standard rate for the decarboxylation react ion of dilute, 7 -6 4.0 x 10- M, 1 4C-tyrosine in the presence of 1.0 x 10 M pyridoxal 2 was determined to be 1.96 10- n moles of C02 min. for a 15 minute x / period. The decarboxylation of 1 -tyro sine in the pre sence of 4c 6 1.0 x 10 - M mimo sine but in the absence of pyridoxal had a rate of
6.08 x 10 _4 nmoles of C02 min. Thi s react ion rate was constant over I a two hour period; whereas with the pyridoxal-catalyzed reaction the rate began to decrease after 20 minutes. The reaction in which mimo sine was first incubated with 1 4c-tyrosine before injecting the ( mixture into the enzyme and buffer + pyridoxal) had a rate of 2 2�74 x 10- n mo les min. for a reaction time of 15 minutes. This / 4 + reaction mixture, 1 C-tyrosine mimosine, was fractionated on the
Sephadex column and by paper chromatography as described in a later Table II. Tyrosine decarboxylation reaction rates with varying mimosine concentrations and constant tyrosine concentration.
Final conc • . of 1 Item of concentration y1co2/15 min. y1C0 /min. l/plC02/min. mimosine/3ml flask of mimosine added 2 A (A] control B6 25. 376 1.692 0.591 -1 - lxlO M mimosine 6.8.670 4. 578 0.218 1.6 7xl0 2 M 60 -2 9xl0 M mimosine 76.2'iD 5.085 0.197 l.5xl0 -2 M 67 -2 7. 5xl0 M mimosine 57.638 3.842 0. 260 l.25xl0-2 M 00 -2 6.0xlO M mimosine 55.200 3.600 0. 272 l.OxlO _2 M 100 -2 5.0xlO M mimosine 52.016 3.468 0.288 8.34xl0 -3 M 120 -2 - 2.5xl0 M mirnosine 42.983 2.866 0.349 4.16x10 3 M 240 - lxl0 2 M mimosine 45. 321 3.021 0.331 1.6 7xl0 -3 M 600
- - 7. 5xl0 3 M mimosine 37. 102 2.473 0.404 1. 25xl0 3 M 800 -3 - 5.0xlO 1. M mimosine . 33.468 2.231 0.448 8.34xl0 4 M 1200 - 2.5xl0 3 M mimosine 32.454 2.164 0.462 4.16xl0-4 M 2400
l\) . Vl . Table.··nr. Tyrosine decarboxylation reaction rates with varying tyrosine concentrations and constant mimosine concentration. Final cone. 1 Concentration of tyrosine added plCOz 15 min y1m2/min l/jllCO�min of tyrosine/3 ml / flask s [s]
-1 _2 l x lO M tyrosine 56. H�l 3.745 0.267 l.67xl0 M -2 60 _2 9xl0 M tyrosine 56. 503 3.767 0 .265 · 1. 5x10 M 67 2 7.5xl0 - M tyrosine 51.832 3.455 0 .289 l.25xl0 _2 M 00 -2 6.0xlO M tyrosine 49. 140 3.276 0 .305 l.OxlO -2 M 100 -2 5.0xlO M tyrosine 52.016 3.468 0.288 8.34xl0 -3M 120
- 2. 5xl0 2 M tyrosine 51.753 3.450 0.289 4.16xl0 -3 M 240 l.OxlO -2 _3 M tyrosine 33.440 2.229 0.448 l.67xl0 M 600 7.5x10 -3 -3 M tyrosine 26. 284 1.752 0. 571 1. 25xl0 M 000
- 5 .OxlO -3 M tyrosine 24. 536 1.636 o .6n 8 .34xl0 4 M 1200 I 2.5xl0 -3 M tyrosine 19.240 1.283 0 .700 4.16xl0 -4 M' 2400
l\) CJ' I f Fig .• 2 Reciprocal plot o tyrosine decarboxylation reaction .rates with variable mimosine conc.,.·cbnstant tyrosine cone. H�Hlttf�HJJi�rWS-1���rmr��f ···• i -1 T i qi,!r 1; ��-11�1 -11I ttI TE tfi, Jj Ijt u ; - 1 1 - t- r 1 - : t: 1 -:-f 1: \-r·: ! 1 ·I - : D' r:Ttt 1 �--rT-±f� 1 :�r,�L�r;t·_JJJ··11��1 �rfl1�IH�t�: i , ,_J___L_, • . � :,_ L• I i . I :wt=�--ju-j_-!. -'-_+'p_J-'--L----'�i-+-LL-l---1 :-+ r1 - 4�-L,::...1--'-"jj -'-� -:��+�ii�� ;�-�8-�:�--·_:_,I -: · ��t�;J-fm�Jt1-�J- "O 1-c·+ f- - f ··· lrTi 1 -:-1 ·• · t-,-; ! ; ' n -, -rn·_ -!iJi1- - j·+-r1•p1t- 11�· +-i1--r,-f1 �rt-r�:r1f1 -+d�·h+ 1
__ ... .. 1 -- + · . · r t � l 1 : r; i-,-f-1-1-��r -•--.--1-F1 -1-1 .-+1- ,1· H �1 1- ·l···"r�· i -, ·ffi
�W_ il+- - - ��fil• lW!iB-lf 1 _ fl--'- , 1 r " ']+J_,_ ; + J - _ii - 1 - , - 1 1 -+ ! .,c - I - . 1 •- 1• · -+ ! _j • - [-• !tf +·- I · • · I fw --- [,1 :��if wrn-1�:�tt�I1lli1JfrJ�tfil_ltiltltiHI1E� l\) .....J o 1. 0 2.0 3.o i.O 5.o ,.o ?, o t.o q.o 10 11 1;. 13 1'1 IS '' 17 1� 1'1 ;..o ,_, ;.1- ll.3 '-'/ ) /_ -3 ,>'£"11 ] t 10 . I ciprocal plot tyrosine decarboxylation reaction rates with variable Fig. 3 Re �f
tyrosirie cone. const ant mimosine cone. [ M:'"".J iJ CoAJs+11�-f. -��I -- ·
i . I I · · - I - . '-'T. ' · - . -:-1 . .. I l1n rtrl t1 I t' ...... 1 � I . I I . I' . l
,, I� Ii' J9 �0 i1 �,_ •\ � ,_y l\) <» 29
section.
Effect of L-Canaline .2.!l Mimosine Catalyzed React ion Because L-cana line inhibits the activity of nearly all pyridoxal-
dependent enzyme s by way of its imine formation with pyridoxal's
aldehyde group , 4g the effect of L-canaline on the rate of decarboxy
lation of 4.0 x 10-7 M 1 4C-tyrosine in the pre sence of mimo sine was
studied. In the absence of L-canal ine the reaction of mimo sine and
1 4 10-1 4 C-tyro sine produced 1.69 x n mole-s of 1 co 2 in two hours. In -2 the presence of L-canaline 4.85 x 10 .nmo les of C02 were produced for the same reaction time. This wa s a 71.3% inhibit ion compared to the reaction in the absence of L-canaline.
Ultrav�
mimosine has two absorption peaks, 226 nm and 304 nm. An absorption
peak at 304 nm was reported by Spenser and Notation. 1 2 Becau se · Metzler demonstrated imine formation between pyridoxal and L-va line, 43
a similar study was conducted using mimosine instead of pyridoxal.
Using 5.0 x 10-5 M mimosine as a reference solution, blanks were run
using the same concentration , 5 x 10 ._5 M, of L -valine and mimosine. L-valine had no ultraviolet absorption above 225 nm. The mimosine blank gave no absorption at all ( see line Fig. 5) . A, A reaction of mimosine with L -valine was observed in the ultra- violet spectra at pH 's 5.5, 7.0, and 10.5. However, the reaction proceeded to a greater extent at pH 10.5 than at lower pH values. 31
� <)! I z a � � .. I I J � � ... ��- � to 0 % >- �j .,;1 I ... 0 ,,.� z � .c: ... � �I � � "' ·�... �� 0 • �.�• �• It � Fig. 5 Ultraviolet Spectra of the Reaction of Mimosine and L-Valine at varying L-Valine concentrations.
Line A is the mimosine blank. The concentration value by each line · is the L-valine concentration after it had been mixed with mimosine. The mimosine concentration for all spectra was held constant at 5xl0 _, M. ,,
,....,.., ...,... Wt1fN lfOllOfltNG, Sl'fON CHAIT NO I� lf«IUI mt1u•11n. 111<..mtflfOI. \.J..) \.J..) 34 Therefore , it is at this pH, 10. 5, that the reaction of mimosine and varying L-valine concentrations is reported (Fig. 5) . When the absorpt ion spectra at various val ine concentrations were compared, two sharp isosbestic po int s were observed at 295 and 240 nm. The � max values for the various peaks at 280 nm were 8.6 :!::. 2.0. Because n-butylamine is soluble in benzene and mimo sine is totally insoluble in benzene and because n-butylamine is a non-aromatic compound capable of forming a Schiff. base, a mixture of n-butyl- - amine and mimosine wa s studied by ultraviolet (Fig. 6). An infrared absorpt ion spectrum was also obtained for this mixture. The spectrum showed a reduction of ·the C pea 1 640 cm- 1 ), and absence of the = 0. k ·( - ( - l o· · · ·H-O-peak 1350 cm ) and the = O peak (1230 cm- 1 ) Fig. 7) , :)c which indicated that a rea.ction had occurred with the formation of an imine· between· mi mo sine and . n-butylamine . By recording _the absorbance of •an old mirno sine solutio0 against a freshly-made solution, it was established that mimosine does react with itself. In Fig. 8, the decreasing peak at 280nm is attr ibuted 2 to the disappearance of· mimosine at pH 5. 5 1 and the increasing peak at 225 nm is assigned to the imine formed between mimosine and mimosine. Sephadex G-10 Column Chromatography The fol lowing values were obtained for the Sephadex G-10 column: the column contained 42. 1 8 g of dry gel, the Wr was_�.O + 0.1 g /g, V 4. 18, 0 1 ml, and V 14� ml . For the calibration of i = V = 4 t = the column using water as the solvent mimosine wa s eluted at 52 to 56 ml Fig. 6 Ultraviolet Spectra for the Reaction .Between n�Butylamine and Mimosine Rev. ine wa s made by interchanging the 1 sample and reference cell�. 36 � N. I ' i ...i i ... � �I I I '1\1 I �· �1 ...� "' I � � � �I I �. 1-i 0 ::IC g ..J .,,... �0 ��:i a; s I - -< on .. .. � � 0 ! � ... '-& "" � "'I. I i . i .i c • c • � a i -H-+.4+-l...;..H-H+��;...,+i�-H--r-1--�-++++--+++-�+-++-rl-H++++t+nrrt-ttt+�·-rrr-rrrr:-t-H-riH-H� +.,-r--��� :c us t � i! � z J � � J: () �Cl " t: (IJ '\. E .:/. 0 Ol m � 'It Ill � ,. ' � § Fig M M • .7 Infrared Spectrum of Residue from ixture of imosine and n-Butylamine in Benzene . WAVELENGTH '.MICRONS) I I 3 4 5 6 7 8 .• 10 ' f ' 12 - . I • ! I I I I . - ,,,, I I -,-- ? . • 1 I ! : . : J- 1 I 10:> : -r--r- . 'I. . -· - - ; '""' _ I - - - .. 1 . I I I _ ! . - ' -- - - -1-- -� I' .!,_ ,_ , _,,_' I ! i , f - - - :..J.:.. -- -;--- · . ,, . . , ... . ,--. . ... 1 ' .u ""i'' ...... I ! · --1-�--- ' , ; ! I · �- · _ 1---; ' _ j_ ._L_ i ___ _ i_Ji _· ___ --i�--l--J�f- - I' ... · _ __ ;_r-;. + . ! ___ , _J_ - + ! � -i-I - -- . · - I . - . -- ·:1 - -- - :� L .!-- I -- . --- - ; - =�-- -�-· -- , - - · - -- 1 : - · , --T- • · · -\ l I I - - I I 'I � ; I . I ! i i I 00 I i , I ! 1 ej · - '. aJ '._J- -r-+- ·- i -1- -. ------1- - -- I .. __ _ ;------· . .. _ _ ' I I I -- I --1--- · ; ; __ I -!� . ! I ·-: - . , - __ ; :-i-- ! _: �_ r , --r--. ··1 - I - _!_ 1--- '. -- . · · - - I - ·· i ' . -:-- , i I ' ' - . ! I I • I i I + - I : ' I ' i 1 _ :__ - t-� ,- I , - ' - 1-- . � I I -- · ______: . : : _ _ ___ -· - ! · _ _ . : i ------I I - : -- -- , - , -- :-· ' j ,. ! • �-·-,- ,_j --1- - - � I 1--�ti · · - : ' :---·i I ---r · -- i- · . - . ·- ' T- -j , ··-- · -�·- , , - -- - i · . . - ---- I , - -. -'-- l ! ·- · I , . I ! TI : . ' _/·_ �· i I : : I� 6D I I :. : ' - I ; I - - -- j'--t I - -ri . --1 _.______- -r - . ' I -- ·--- i - -·1 �-l- --- -· - -- ! ! I\ I �! �! ...... - i, ·:l - L- l L_L_ , -�f-r- :- j T , .. ·-· j__ ! - --'. - · 1 - - ! ; .. i----i-+------1·· · 1 ·------· . , - i -- 1 r r- i 1 , l I i T I - I [ - . _ _ _ _ . -- ' _ .... __ __ _ ;--__ _ ,: --, -- - -, - -·:-- -1 _:_ .. ; : . -. ; ; j ; . i i ; · · �; : · - · I I 1 · --· . +- : : , - - , --T- r I . - : • -1 . -', -rI I 1--�l -- . . . . i,-- .. - 1- --- .. - . : . I · --- .. .. · I +-i · . - I l - ·- - ! ...... · · ----fI � y- ' -- 1 ,.,_ . : . ··+ . 1-I --0.. · ·i·-- - -- ·- �- . ... . I I ,: - :- i ' · - -�-- I I , --1-· - �1 ---��< . _L;. 1 •I - : . • i I I 1 I I . :.... -- --1- - . -·- �'-+-1- . , . ·-•- •1 , -r--t - [�t 11 : : -1 --; -l . _ . -1------r ' i I __ _ : __ -·------· · - __ __ --- · . - ! · I i -· - : -- 1 : -- ! I ' 1-. -�- -1 !1 -- .· ,: . -- - [ ._ . I .. . H··- -- --r---- -: -- --- ; . .. · i- · - ' · , 1-- --- :-- "-- -i• • • - I ·- · - --T- I : l=J 1 I ; --· i- I - r , . . ---� : . ! 1 20 . ': t . - 1-- -·- I - :J : ! ' � I -1 ---i. · l ----� 1-- ! I ' - , -- - -- ______I -. ------. ______i - ______... I ' I • - I --I r-- - I I I ! I ! ' _ : , - rI - -� - - · ' --�--- [ _J__ : - , .1 :- -T-- _• _ �·:-- _ , ..-1------:--- ,- · - _ . ---f- =R--1- --i------, - - r- ! ! !_ _ I + I I : S ! 20 i --+ . 1 T- . I --- . __ . . l • : . *! I . 1 .. ...' - -·' --:-· . : L -- , - -- 1 - --- i' -· --t-·- , �!L . - ·-- 1 : · · !-- 1- i-· I ' --: - ·-�---·. 1 + . I j -c.r:-�= 'I I! ---f-- • : . • -· I . • -�--- j -- I I ---l-- !- - - ·r- J : I 1 . 3500 3000 2500 2000 1800 1600 . 1400 1200 1000 800 WAVENUMl'.-ER (CM ') ( w -...::z I Fig. 8 Ultraviolet Spectrum of Mimosine-Mimosine Reaction The absorbance of an old mimosine solution (5 da s y ) 4 at lxl0- M wa s recorded against a freshly made solution also at lxlO -4 M. Both samples were at pH 5.5. .. ,. -- . 39 ' ...� I')' Il . ;il -; � l J I � � . !J �o � � QI ! ...� ! > �� Cj +I I 1• l � � ,. � I I� 2 d 0 l \ � � 2: � -� � "'·1 �0 � � > � l ill � � I � .,, ,,,I I I i � I I 0 = � �1 1� l �n� n : � � 40 and 1 4c-tyrosine was eluted at 77 to 81 ml (Fig. For the cali- 9). bration no radioactivity was detected except between 73 and 83 ml . 4 When a sample from the reaction mixture of 1 C-tyrosine + mimosine + enzyme wa s applied to the Sephadex column a radio active peak was detected between 48 and 51 ml . This peak could be assigned to the irnine of mimosine and 1 4c-tyrosine . A smaller peak was observed between 64 and 68 ml. This peak was not identified. Paper Chromatography The Rf values obtained for standards, reaction mixtures, and radiochrornatograms are reported (Table s IV and Considering all V). the compound s and possible products in this study only mimo sine gave a bright pink or red spot with the diazotized sulfanilic acid - NH40H · 1 1 4 · spray. When using a samp e o f th e c - t yros1ne + m1· mos1n· e + enzyme reaction mixture described previously, it was noted that after 24 hours of reaction time , two equally intense peaks were observed on the radiochromatogram: one at Rf= O .47, tyro sine, and one at Rr= 0.61, tyramine (24 hour, Fig. 10) . This proved that tyro sine wa s decarboxy- l ated to tyramine by tyro sine decarboxylase in the presence of mimo - sine. As the reaction time increased the tyrosine peak decreased and the tyramine peak increased (36 hour sample, Fig. 10) . At 48 hours the reaction is essentially complete and only one peak is observed, tyramine at R 0.61. In the absence of mimo sine no COz or tyramine f = were produced. This indicated that rnimo sine may act as a coenzyme for the tyros ine decarboxylase reaction and does so in the same manner as pyridoxal. This continuation of the mimosine-catalyzed decarboxylation 41 Fig. 9 Sephadex G-10 Column Fractions io.o 9.o � ' r l\ s. o I i I I \,\ · ..-·o \ \ �r-1 7 .o l x I I 6 JC I N 5 . • I I I o I 0 ' }\ r-1 ' \ · x 4. .o I :E I 0... ' u J . o \ . I I I \ I I I / , \"- ,...., ______40 50 60 70 80 90 Peak A is rnimosine as measured by ninhydrin. �eak B is tyrosine as mea sured by the amount of 1 4C pre sent. B' and C are the radio 1 4 active peak s from a rnimosine + C-tyro sine + enzyme reaction mixture applied to the column. represents the calibration peaks and ------represents the reaction mixture. 42 Table IV. Paper chromatogram Rf values for known compounds detected with diazotized sulfanilic acid -NH40H spray. Compound Rf values L-Tyrosine 0.48 :t. .02 C-Tyrosine 0.48 .02 J. '+ :t. Mimosine o. 22 :t. .02 Tyramine HC l 0.57 + .02 p-hydroxylphenylpyruvic acid 0.82 :!:. .02 Dopamine 0.38 :!:. .02 43 Table V • Paper chromatogram R f values for known and unknown component s . of . tyrosine decarboxylase _ reaction mixture. Compound Condition or proof R f values 0 Mimosine Known sample applied to 0.21 2:. . 1 reaction mixture spot over spot ( ) Tyramine HC l Known sample applied to 0.61 + .01 react ion mixture spot over spot ( ) 14 C tyrosine Known sample applied to 0.48 :!:_ .02 reaction mixture spot overspot · a ( ) Tyramine Radioactive spots identi- 0.61 + .01 fied by overspotting with known and spraying it with .diazotized sulfanilic acid NH40H spray. a 14 0 C-Tyrosine Radioactive spot s identi- .47 :!:, .02 fied by overspotting with known and spraying it with diazotized sulfanilic acid- NH40H spray. a Unknown imine Radioactive spot turned red 0.31 + .01 ( ) when oversprayed with diazo- tized sulfanilic acid-NH40H spray a Radioactive spot s were detected by the Radiochromatogram Scanner. 44 14 Fig. 10 Radiochrornatogram of timed reaction mixture of C -tyrosine, mimosine, and ehzyme. ·· - · ··· · ------·· · --- - �·------:-- - ... ------:- ; : - .. ' � -· : - _.:,__ · -· - -· I: T . - --· -� -· - - ,'; - · - -- · : �, t - - ·- - · -- - ...: ------: : - � . - - - .-- -- · -- ··• - - -- - �. . . ··-· --� -- -· .. · : · .. · --·!- - · - · ...__ - · 2 ------4- - - .. -J -- - · - - \. h- ___ _�_ : ... · -·-: --- our ---�--- - . . - :-- --·· ! � . - -· ·-: . .. -----� ! ---··�· I - '- • ·· - · -- - ·-- - • -· - - • ..• . -- ·-+------· - ··--'· .... t-, t-, -:-i - '.t - _ _ - L � --- -�------�------�: ------�v!fi;./-.Jt� �� � - -- ·-· ·· --· -· ---· -· - - - - -· - ·· -·- · -� - -- •·· __ ..:_·-- +· ···· : : -· - +- -�· � ···- --··· -----· ·--····- -· . ··- �---- ···• -- - - - ·· ·--· - �-��.:.L� ,:, -- -- -··- _· _ _ ___ · ·· · Mt __:__�_..:___:_... .._ •. i --.·�·-·---+-·- · I -· -· ··· ------·-·-·-·· -� c.:..·.,-"w.;; 3 The full scale linear range of the above ' graphs was lxlO counts/min. The is tyro sine and the B eak is tyramine. A pe�k p 45 o� :tyrosirie over long period s indicated that mimosine wa s being r�used in the formation of the imine with tyrosine and that a constant turnover: mimo sine + tyrosine to imine . and . imine to mimosine + tyramine. F g 11 s ows the chromatogram of the reaction mixture of i . h mimosine 14 + C-tyro sine + enzyme and the chromatogram of the Sephadex column fraction number ml from peak C in Fig. 49 ( 9). 46 Fig. 11 Paper Chromatogram of. Reaction Mixture a nd Fract ion 49 from Sephadex G-10. �ryamine 0 0 Tyrosine 0 0 0. 0 Mimosine 5 1 2 3 4 No. � radiochromatogram of mimosine + tyrosine + enzyme reaction. No. chromatogram of mimosine tyrosine enzyme reaction sprayed · 2 + + with diazotized sulfanilic acid-NH40H spray. No . 3 fraction No. 54 from Sephadex column, sprayed with diazotized sulfanilic acid-NH40H spray. No . 4 radiochromatogram of fraction No. 49 from Sephadex column. No. 5 chromatogram of fraction No. 49. 47 DISCUSSION It was shown by the Warburg work that tyrosine decarboxylation in the presence of mimosine was not further increased by addition of 2+ Mg in excess of the mimosine concentration, thus indicating that the tyrosine decarboxylase + mimosine system cannot be further activated by metal ions as are some enzyme systems. The Warburg work also proved that under in vitro conditions the complexing of mimosine to pyridoxal is important and can stop --the reaction completely due to a lack of coenzyme. It was also observed that mimosine was not a substrate for the tyrosine decarboxyla se reaction. r from the kinetic study , the curve with tyro sine concentration, . as the variable Fig. 3) was very similar to the curve with St , ( mimo sine concentration , A , as the variable Fig. 2). The two graphs , t ( have the same two inflection points: one where the �yrosine concentra tion is equal to the mimo sine concentrat ion an d the other where mimo- sine becomes limiting . Parts B and C for each graph can be shown to fit characteristic curves for the effects o f activator limitat ion. as s.hown by John . 2 Reiner. 5 Part A for each graph was attributed to the effect of pyridoxal on the enzyme and on the com plexing of pyridoxal with mimosine. The extent of the complexing of pyr idoxal with mimosine is hard to establish and for the graph with varying mimo sine concentration it is impo ssible to determine . It is this effect 0£-�pyridoxal that makes the graphs not completely superimpo sable as they should be according to Reiner ' s mathematical treatment for the act ivation of 52 sub strate. Reiner showed that when l/At � l/St the plot is hori2on . tal · and · when / > /S l At l t the plot is a line of the convent ional type , part B. The point at which the cu.rve changes is at l /A l/ · t = �t For both of the graphs studied, an inflection point occurs at this point , l /A l/S · t = t If one considers the B part of the graphs as the conventional type for the act ivation of substrate, the solution for the curve is l/v ( l/V) ( l Ki/A ) with a slope of_ K /V and an intercept of l/V.55 = + t 2 { See appendix). If one has an activation of a substrate, l /V or V for both graphs should be the same. For the system studied l /V for part B of each graph was exactly the same. The deviation in the slope s or K 2 is again attributed to the pyridoxal interaction with mimo sine and can be ex plained as follows. If the pyridoxal does interact with mimo sine, then the amount of free pyridoxal will vary depend in� on the amount of mimo sine present. If pyridoxal causes a conformational change in the enzyme then the amount of free pyridoxal would affect the rate of �inding , and thus k 2 k3 ( , A). the valu� of k2 E + Sa � Ca Ca -+ E + P + k_2 k 2 - + k3 From appendix K2 = This effect of pyridoxal on K 2 can be shown with the two graphs. I f one assumes that the interact ion between pyridox�l and mimosine were constant for the case in which the mimo sine concentration was �onstant (Fig. 3) , then the slope of part B of this graph can be 49 considered . the normal one for the re action. Comparing this to part B where the co ( ncentration of mimo sine is less than the tyrosine concen trat ion of the graph Fig. 2) in which the concentrat ) ( ion of tyros ine is constant and mimo sine ' s concentration varied , and knowing that part B of Fig. 2 has less mimo sine present to interact with pyridoxal higher pyridoxal mimosine ratio , then the amount of free pyridoxal ( / ) 2 in part B of Fig. wo uld be higher than in the comparable region of the normal grapt, Fig. 3 , assigned above . ( ) I f the pyridoxal does cause a conformational change and if the conformational change is concentration dependent , then the more pyridoxal present the more often a conformational change could o ccur. As a result, because there is a higher effective pyridoxal concentra tion in part B of Fig. 2, the k2 would be higher and Kz lower than for Fig. 3 . This wo uld mean that the slope (K2/V) would be smaller for part ·of Fig. compared to part B of Fig. This is in fact B 2 3. what is observed. Therefore, if K2 is different for each concentra tio pyridoxal, then r see appendix wo uld differ from r n of free a ( ) s and the two graphs would not be superimpo sable but would have the same V or intercept . Therefore, because both graphs had common inflections at l l S and· because pert B of both graphs the intercept or l V /At = / t in / was exactly the same and the variation in slope can be attributed to the mimo sine-pyridoxal interaction, it was concluded that mimosine sub strate in the tyrosine decarboxylase causes an act ivation of the reaction. One possible mechani sm for this activation is by imine formation between mimosine and tyro sine. The ultraviolet study suggested that imine formation can occur between mimosine and various amines. In the ultraviolet spectra of the mixture of L-valine and tyrosin2 two isosbestic point s were obtained, 295 and 240 nm. The intensity of three absorption bands also vary with the concentration of valine present, (Fig. 5). The two decreasing bands, 310 and 232 nm, were assigned to mimosine (Fig. The increasing band , 280 nm, may 4) . correspond to one reported by Metzler43 at 280 nm for the imine between pyridoxal and L-valine. Because the mimosine peaks, 310 and 232 nm, are on one side of the isosbestic points, it wa s concluded that mimosine is in equilibrium with some co�plex of mimosine. It was observed that as the concentration of valin� is increased in· the reaction mi xture the amount of free , uncomplexed mi�o sine pre sent decreased and the absorbance on the other side , 280 nm, of the isos bestic po ints increased. Therefore, it wa s concluded that mimosine was in equilibrium with a complex of mimosine and L -valine. Further on that mimosin wa s being consumed by the ,formation of the indicati � mimo sine-valine complex was obta ined by a reversal of the reaction cell and the reference ce ll in the spectrophotometer. If the re ference cell has mo re mimosine present than the reaction cell a pos�� ive absorption will be observed ( the reaction was started with an equal the re concentration of mimosine in the reaction cell and in refe nce cell). A po sitive absorption was obtained in all cases of rever sal of cells. 51 It should also be noted that for the imine formation between pyridoxal and L-va line a similar increa5ing p9ak wa s observed at 43 2$) nm and an isosbest ic point at 296 nm wa s reported. Similar points are also obtained for the mimo sine -valine study. The max � 280 values for the peaks at nm were small, 8.6 .:!:, 2.0. These values are consistent with tho se proposed for i�ine formation. 53 The longer wavelength than usual for the imine formation is attributed to the additional interact ion of the K- and .B"'! band s on the R-band ( n- ) 54 ,;$ of the -C = N- bond. The n-butylamine - mimosine reaction was also assumed to exhibit an equilibrium between mimosine and a mimosine-n-butylamine complex. The infrared spectrum of this reaction mixture (Fig. 7) indicated a 1 reduction of the C = 0 ( 1640 cm- ) absorption peak and the absence of the ...H-0 cm- 1) absorption peak and the �� = O_ (1230 cm- 1 ) -0- (1350 = ·absorption peak. Because of �he loss of these. peaks it w9 s concluded that the complexing of mimosine with n-butylamine takes place at the 0It -C- of mimos�ne and is �n the .fo.rm of an imine ( -C = N-) . Because mimosine has an cX.-amino group in its .s ide chain, mimo-- sine should react with itself, if the imine formation occurs through the ketone group of mimo sine. It was observed that mimosine does react with itself ( Fig. 8). Further ev idence for the imine formation between mimo sine and tyrosine was obtained from Sephadex column separation work. Because of use of pyridine in. its preparation the S.ephadex G-iO column had an increased aromatic adsorption affinity along with its molecular- 52 sieving properties. When the reaction samp le of mimo sine + 1 4C- tyro s ine + e nzyme was applied to the column, a radioactive peak was obtained in the 48-51 ml fractions. Because the larger compounds come off first , it was concluded that the peak, 48-51 ml fraction, represented a reaction pr oduct or an intermediate of molecular weight range between 198 and x 106 . Also because the peak at 49 ml 2 was radioactive it had to represent so complex of 1 4c t yros . ne. The me - i paper chromatogram of this fraction (49 ml) showed a red mimo sine spot, a radioactive tyramine spot, and a new pink radioact ive spot. The pink color of this new spot wa s attributed to the mimo sine moiety and the radioactivity wa s attributed to the tyramine moiety . Because imines are easily hydrolyzed it is not surprising to see the tyramine, mimo sine and mirnosine-tyramine imine spots on the paper chromatogram of an aqueous sampl e. Therefore, this new fraction. corning off the sephadex column was probably the imine of mimosine and tyramine with a theoretical mo lecular we ight of 317. 4 By spotting various timed reactions of mimo sine + 1 C-tyrosine + 4 enzyme pyridoxal , it was observed that 1 C-tyro sine is degrad d ( no ) e 4 Fig. ) to tyramine and 1 C02 ( see 10 and that given enough time all of the tyrosine is degraded. Mimosine was abserved to stimulate the enzymatic decarboxylation 4 of tyrosine at a very slow rate, 6.08 x 10- n moles of CO z/min. , compared to no reaction in the absence of mimosine. This reaction -- rate wa s, however, constant for up to two hours. The pyridoxal- catalyzed reaction exhibited a more rapid rate of decarboxylation, 53 1.96 -2 x 1 0 n moles of C0 2/min. , during the first 15 minutes, but the reaction essentially stopped after 30 minutes. The constant reaction rate for mimo sine-stimulated decarboxylation is attributed to the fact that the imi.ne of mimosine with tyramine is unstable and is very easily hydrolyzed to its amine and ketone products. Thus mimosine is constant ly being regenerated. This instability of the mimo sine imine explains why it is so hard to isolate. Further evidence that the carbonyl group of mimo sine is quite reactive wa s that L-canaline strongly inhibited mimosine 's catalytic 4 effect on a mimo sine + 1 C-tyro sine + enzyme reaction system. L-Cana- line has been reported to in hibit many pyridoxal-dependent enzymes by way of oxime or Schiff base formation of the canal ine with pyri 4g doxal phosphate. Structurally mimosine has the main functiona l groups requ ired by " pyri d oxal to catalyze the non-enzyma t1c. reac tion: .39 a carbonyl group ortho to an hydroxyl group and a carbonyl group in conjugation with a hetero c yclic ring nitrogen. Because the 2-methyl group is not e s sential for pyridoxal act ivity and can be replaced by a hydrogen 45 atom, the functional resemblance of mimosine to pyridoxal becomes 44 46 more evident . It has also been shown with two enzyme sy stems ' that it is the phenolic hydroxyl group and the pyridine nitrogen that are required for the binding of pyYidoxal at the active site of the enzyme s mimosine also ha s these groups . Mimosine -could bind to ( ) 47 enzymes by several of the methods propo sed by Snell. Therefore, structurally it is not surprising that mimosine can form an imine 54 with amino acids as does pyridoxal. 2 Data from earlier work by Tung et al. 0 showed that the L-DOPA 2 decarboxylase Teact ion was also catalyzed by mimosine. 7 in --- et al. · L . showed an increased rate of decarboxylation in the presence of L-[X)PA a low concentration of mimosine. The latter observation may now be explained by mimosine's activation of the in the same manner as IXJPA mimosine activates tyrosine in the tyrosine decarboxylase system. The 2 observation of Tung et al. 0 can be explained by mimosine 's substitu- tion for pyridoxal as it was observed in the present studies with tyrosine decarboxylation. The receptor sites of many enzymes which utilize amino acids as substrates can be depicted as having two binding sites and one active site. The binding sites can be designated as $pecific or non specific. 56 In pyridoxal-requiring enzymes one of the binding sites is for the pyridoxal moiety and the other is for the substrate . The two binding sites cause the pyridoxal- substrate complex to place the carboxyl group of amino acids in juxt apo sition to the site of chemical change, the active site. I f the pyridoxal binding site is nonspecific, then mirnosine becau se of its similar groups could fit into this bind- ing site. If this substitution does not alter the orientation of the carboxyl group of the amino acid then the reaction can take place. If mimosine does affect the orientation and once bound to the binding site cannot be released, then the enzyme system is inhibited . In the tyro sine decarboxylase enzyme system it is suggested that a conformation change mu st take place , caused by pyridoxal, before the - 55 mimo sine-substrate complex can fit into the receptor site. This con formation change is thought to facilitate the accessibility of the receptor site to the preformed mimo sine-sub strate complex, Once the complex is there and if the orientation is correct, the mimosine substrate complex can undergo decarboxylation of the tyrosine. Because of this difference in specific ity at the pyridoxal bind ing site and because the resulting orientation of the active group at the binding sites may change from one enzyme system to another, the varying effect s of mimosine on dif ferent enzyme systems can be ex plained. 56 SUMMARY A number of experiment s suggest imine formation can take place between mimosine and various amines. Imine formation between mimosine and L-valine was suggested by the ultraviolet · study with mimo sine and varying concentrations of L-valine. The separation of the reaction mixture of 1 4C-tyro sine + mimosine + enzyme by the Sephadex column and subsequent analysis with paper chromatography indicated that an imine is formed between mimo sine and tyrosine. The infrared spectrum of the n-butylamine + mimosine reaction and the inhibition of a mimo sine-stimulated react ion by L -canaline showed that the imine formed between mimo sine and various amines takes place through mimosine 's ring carbonyl. The significance of the imine formation becomes important when one couples it with mirnosine 's similarity to pyridoxal. Because mimosine has the same main functional groups required by pyridoxal for catalyzing enzymatic react ions and for binding to enzymes, it is not surprising that mimo sine can stimulate enzymatic react ions. This wa s supported by the fact that mimo sine does Gatalyze tyrosine decarboxylation. The structural similarity of mirnosine to pyridoxal may also account for the various effects of mimosine on different enzyme systems. The k!netic study indicated that mimosine causes an act ivat ion of the substrate in the tyrosine decarboxylase reaction. It was concluded that this act ivation of substrate is a result of imine formation between mimo sine and tyro sine and of t�e nonspecificity 57 of the pyridoxal-binding site. The kinetic studies indicated that pyridoxal pho sphate is required for the stimulation of tyro sine decarboxylation by mimosine. Pyridoxal phosphate may cause a con formation change in the enzyme so that the preformed imine between mimosine tyrosine can fit into the receptor site and undergo + decarboxylation. without any further steps. Therefore , I hypothe size that the act ive ring carbonyl group of mimosine and the simil arity to pyridoxal are responsible for the inhibition or act ivation of enzyme systems by mimo sine . 58 APPENDIX Scheme for Activation of Substrate S 1 a 2 + A � S E + Sa � Ca - 1 -2 Ca E + A + P where S = substrate 4 A = activator E = enzyme P = product Sa = activated substrate Ca = enzyme complex of the activated su bstrate Then with the conservation of enzyme , = E + Ca, and by solving Et the steady-state equation for Ca, we get Ca = E t Sa/(K2 + Sa) where Kz = ( k z+ k ) • _ kz This can,r' be shown to be the same as _ (K2 + Sa) = VSa/(K2 + Sa) lv - V Sa v K2 + K2 = l ( 1 + ) Now, when At St then Sa = At • Then 1 = At V At < v --v�A-t� and the slope would be K2/V and the intercept l/V. Then by assuming conservation of A and S, A A Sa + Ca and t � + St = S + Sa + Ca, and using the relative velocity, r wh ere r = C a/Et and k3E = V ) . We re-solve the steady- (note r= k3Ca , k3Ca = v, t state equatK;qion and get At = rEt + K2r/(l-r) + K 1K2r/ [C 1-r)( St-rEt)-K2r] where K 1 = (k_1 + ki )(k1 +k2)• •' . 59 One of the limiting values of r, for which becomes very large, At can be determined by setting the denominator of the third term equal to zero and solving that quadratic for r. 2 r 1/2 (1 + ( S + K2 )/E + ( S + K2)/E -4 s /E a = ( t J :!:. "� t J t t Doing the same for St , we get : ) r 1/2 + + K2 )/E + ( A + K2)/E -4A1/E s = ( [i (fit J :!:. Vr1 t J t Since r r/k E , then r V /K3E , r V5/k E · The partial ) = 3 t a = a t s = 3 t s aturation rates are therefore obtained by multiplying ra .·and r5 by :l V /2 k3 E + S + K2 ) - (E + S + K2) - 4 St E k,Et a = t t v t t t 1 U 2 } V5 1/2 k3 E + A + K2) - V(Et + At + K2 ) - 4 A · = t t t E{] Va and V 5 being relITative velocities at partial saturation. 60 REFERENCES M)RRIS, Repart. Pharm. 9, 364(1897 ). 1 . , E. Y. HOSAKA and J.C. Hawaii Agr. Expt . Sta. 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