HISTIDINE AND CYSTEINE CAN ENHANCE THE METABOLIC REACTION RATES IN
THE TCA CYCLE AND SOME AUTOTROPHIC PROCESSES: GENETIC CODE WORLD
Mikio Shimizu*
Institute of Space and Astronautical Science
Yoshinodai, Sagamihara, Kanagawa Prefecture, 229-8510*Prof. Emeritus
Present mailing address: 1738-6, Kohgasaka, Machida, Tokyo, 194-0014;
Tel: 042-723-7584; E-mail address: mshimizu55@ybb.ne.jp(Received 8 March, 2007 Accepted 7 June, 2007)(Abstract)
It was found that cysteine or its ester canenhance various reaction rates in the TCA cycle
such as aconitase, isocitrate dehydrogenase,succinate dehydrogenase, and fumarase and that
histidine or its ester can enhance those such as
succinate CoA synthetase and citrate synthase.
Histidine or its ester can also enhance the reaction
rates for carbonyl aspartate dehydrogenase, 2-keto3-deoxy octonate aldolase, and acetoacetate
decarboxylase. These findings for the origins ofmetabolism as well as those for the origins of thegenetic code and the central metabolic pathway in
our recent publications [2-4] suggest that life would
have started from a system of amino acids andanticodonic tri-ribonucleotides. Some possible
routes to the contemporary biosystem from this“genetic code world” were discussed.
Keywords: amino acid enzyme, anticodon
ribozyme, TCA cycle, autotrophe, genetic codeworld
1. Introduction
The central metabolic pathway of biosystem isconsisted of two major processes: (1) the energy
acquiring pathway such as Embden-Meyerhoff
pathway (or non- phosphorylated Entner-Doudorff
pathway in thermophiles) and (2) the TCA cycle[1]: The former cleavages glucose (C6 sugar) to
pyruvate (C3 sugar) and then to form acetyl CoA.
The latter starts from the reaction between acetyl
CoA and oxalacetate to form citrate, which isconverted to various organic acids successively, andfinally to oxalacetate, the first substrate of the cycle.
The cycle produces many starting substrates
converting to amino acids, nucleotides, and lipids
later.
The results in my previous letter [2] showedthat histidine acts as pyruvate carboxylase (ligase)to form oxalacetate from pyruvate and carbonate
with ATP , as oxalacetate decarboxylase (lyase) tocleave oxalacetate to pyruvate and CO2, and as
glucose-6-phosphate isomerase (isomerase) weakly
but specifically. GpUpG, the anticodon of histidine,
had also a similar enzymatic activity stronger by
one order of magnitude than that of histidine, whichshowed a specific relationship between an aminoacid and an anticodon, namely the stereochemical
theory of the genetic code.
I have also shown in a separate letter [3] that
not only histidine but other single amino acids suchas cysteine, glutamic acid (or aspartic acid), lysine,and tyrosine (and their cognate anticodons, too) can
act as the specific catalysts of various metabolic
reactions including transferase and oxido-reductase
in addition to the three classes of enzymes discussed

Viva Origino 35 (2007) 73 - 81
© 2007 by SSOEL Japan- 73 -

in the previous letter [2]. Furthermore, it will be
studied in another paper [4] that two single aminoacid, histidine or cysteine, can convert glucose to
pyruvate and then to acetyl CoA and ATP (via nonphosphorylated
Entner-Doudorff
pathway)
catalytically.
In this report, I shall attempt to check that all
reactions in the TCA cycle can also be enhanced byhistidine and cysteine. Furthermore, some otherimportant metabolic reactions for autotroph of thesystem will be investigated. Photometry will be
used to find the catalytic capacity of these aminoacids, measuring the initial velocities of thereactions. Confirmation of the formation of thereaction products will be attempted using cold thinlayer chromatography (TLC) and high performanceliquid chromatography (HPLC).
2. Materials and Methods
2.1. Materials
All biochemicals were from Sigma. Someamino acids exhibit rather low solubility. We foundin preliminary experiments that the esters of aminoacid, which were blocked at their carboxyl groups
and are much more soluble than the amino acids
themselves, could act as the substitutes ofcorresponding amino acids, while N-acetyl aminoacids , which are blocked at their amino group,
usually lacked rate-enhancing activity. We
employed the esters when high concentrations ofamino acids were required to detect weakinteractions. For leucine, isoleucine, methionine,phenylalanine, tyrosine, and tryptophan, we usedtheir methyl or ethyl esters, or t-butyl ester for
glutamine. Their rate-enhancing behaviors were thesame as those of the corresponding amino acids at
an equimolar concentration. Some of the aminoacids such as lysine, arginine, aspartic acid, andglutamic acid are of salt form. The counter ions of
the Tris and acetate buffers are hydrochloride andsodium, respectively.
2.2. Enzymatic Assays
We examined all twenty proteinaceous aminoacids to determine their rate-enhancing activities in
the metabolic reactions one after another. At first,
the specific reactivity was measured using thesensitive spectrophotometry. The substrates weresolved in the buffer solution and mixed successively,
adding the amino acid- buffer solution at last.
Absorbance of the above mixture solution poured in
the cell of the Beckmann DU 65 spectrophotometer
was measured periodically. The experiments were
performed at room temperature (25 0C). In thefollowing photometric assays, 100 µl of the reaction
mixture, whose final concentrations were specified
in the figure captions of each reaction were used.

Viva Origino 35 (2007) 73 - 81
The above spectroscopic assays were performed as
described in the standard data handbooks [5, 6].
Each assay will be described in the figure caption,
as well as the adopted reactant concentrations.
In the following reactions, the concentrationsof amino acids are much higher than those of thereaction substrates. Consequently, almost all thesubstrates combine with the amino acid catalyst at
first, in contrast to the usual catalytic reaction where
enzymes are almost fully occupied with thesubstrates quickly before the final catalytic reaction
proceeds.
In the cold TLC, the two dye were used : (1)silver nitrate with a back colour of brown ( 1 Msilver nitrate aqueous solution 50% and 1 N NaOH
solution 50% ) for figures 2(b), 5(b), 6(b), and 7(b)and (2) 3-5-dinitrosalitylic acid with a back colourof yellow ( 0.5% 3-5-dinitrosalitylic acid in 4N
ammonia aqueous solution ) for figures 3(b). The
reaction products were usually developed in thebuffer of 70 % n-propanol -30 % 2 M ammonia
aqueous solution, except the hexokinase case,spotting 0.2-1 µl of the reaction mixture on thecellulose plate. The Rf’s of the spots were
determined using authentic samples, which wereindicated by arrows in each figure. The cold TLC
has two factors, Rf’s and colour, and is an adequate
method to observe the formation of the reaction
products such as citrate, isocitrate, and malate
which have no absorption in visible and ultraviolet
range and are difficult to detect by the HPLC
method.
The confirmation was also done using HPLC,
where the reaction mixture was applied into a RPC
5 column (Nishio Tech. Co.). The used wavelengthis 260nm. The elution buffer started from pH 7.8
Tris 1 mM buffer with a linear gradient of 20 % of
0.1 M sodium perchlorate solution in 24 minutes.
The positions of the peaks of various compounds
were determined using the authentic samples.
Disposable tips and tubes were sterilized at
190 0C and 1.5 atm. for 10 minutes.
3. Results
3.1. TCA cycle
Here, we study amino acid catalysts in the
TCA cycle, one after another, in the presence ofvarious coenzymes such as NAD, FAD, and CoA.
Byproducts in this cycle could be used for the
production of amino acids as well as nucleotides.
Figure 1 summarize the scheme of the studied
reactions [1]Aconitase,isocitrate
dehydrogenase(abbreviated as DH later), alpha ketoglutarate DHtype activities of cysteine: These three reactions canbe enhanced by cysteine or its ester (Cys or CE).
Figure 2(a) and Figure 3(a) illustrate the increase of
NADH absorption at 340 nm for aconitase andisocitrate DH, respectively. Nineteen other aminoacids (19 aa), or the reference ( which replaces theamino acid buffer solution by the buffer solution)
did not result in the decrease of absorption.
Figure2(b) depicts a TLC plate showing the cisaconitate COOHCH2C(COOH)CH(OH)COOH spot
(left) before the reaction and the formation of anisocitrate
COOHCH2CH(COOH)CH(OH)COOH
spot near the origin of elution (right) after thereaction. Figure 3(b) illustrates the appearance of analpha-ketoglutarate COOHCH2CH2COCOOH spot
after the reaction (right), while an isocitrate spot isseen on the left plate before the reaction. The
reaction scheme of alpha-ketoglutarate DH issimilar to that of pyruvate DH, which will be

Figure 1. Reaction scheme for the studied metabolic
reactions.

Figure 1. Continued.

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Figure 2(a). (ACO) Time sequence of aconitase
activity of cysteine ethyl ester (CE) using the coupled
enzymatic method with isocitrate dehydrogenase byuse of the absorbance at 340 nm of NADPH . Arel 340
means the relative absorbance at 340 nm to theabsorbance at t = 0. Final concentration: pH7.8 Tris
buffer 100mM, cis-aconitate 1 mM, NADP+ 0.3 mM,
Porcine Heart isocitrate dehydrogenase 3 units andcysteine ethyl ester or 19 other amino acids, 10 mM.
Nineteen other amino acids and the reference showedvery low activity due to the spontaneous reaction.

Viva Origino 35 (2007) 73 - 81

Figure 2(b). (ACO) A cellulose TLC plate showing
the disappearance of cis-aconitate (cisaco) andappearance of isocitrate (iso) after the reaction timeof 1 day (right) in the presence of cysteine ethyl ester(CE). In the left figure, the spots for the reaction
mixture at zero time were shown. This is the same
below. The top and bottom of the figure indicate theend and start of elution. The positions of authentic
samples were indicated by arrows: CE for cysteine
ethyl ester, cisaco for cis-aconitate, and iso forisocitrate. Final concentration: pH 7.5 Tris buffer 300
mM, cis-aconitate 50 mM, and cysteine ethyl ester
200 mM.

Figure 3(b). (ISOD) A TLC plate showing thedisappearance of isocitrate (iso) and appearance ofalpha-ketoglutarate (keto) after the reaction time of 1
day in the presence of cysteine. Final concentration:
Tris pH 7.8 buffer 100mM, isocitrate 50 mM, NAD+
20mM, cysteine 100mM.

Figure 4(a). (SUCS) Time sequence of succinyl CoA
synthetase activity of histidine methyl ester (Hm)observing the absorbance of succinyl CoA at 230 nm.
Final concentration: pH 7.8 phosphate buffer 50 mM,
succinate 20 mM, CoA 10 µM, GTP 10 µM , andhistidine methyl ester or 19 other amino acids 5 mM.

Figure 3(a). (ISOD) Time sequence of isocitrate DH
activity of cysteine ethyl ester (CE) observing theabsorbance of NADH at 340 nm. Final concentration:
pH 7.8 Tris buffer 100 mM, isocitrate 100 mM,
NAD+ 100 mM, cysteine ethyl ester and 19 otheramino acids 100 mM.

discussed in another paper [4] in details.
Consequently I shall not attempt to study thisprocess here.
Succinyl CoA synthetase- type activity ofhistidine: For this activity, I checked the reverse
reaction to form succinyl CoA from succinate, CoA
and GTP. Figure 4(a) depicts the time sequence of
succinyl CoA COOHCH2CH2COSCoA absorption
at 230 nm in the presence of histidine methyl ester(Hm). Nineteen other amino acids and the reference
showed no change. Figure 4(b) illustrates a HPLC
diagram showing the formation of succinyl CoA
from CoA and succinate by the succinyl CoA
synthetase-type activity of histidine methyl ester(Hm) after 1 hour. PiP and succinate COOH
CH2CH2COOH have no absorption at 260nm.
Succinate DH, fumarase, and malate DH-type
activities of cysteine: All these reactions arecorrelated with cysteine or its ester. Malate DH will
- 75 -

Figreu 4(b). (SUCS) HPLC diagrams of the reaction
mixture after 0 and 1 hour for the succinyl CoA
synthase activity of histidine methyl ester (Hm).
Arrows show the positions of the molecules
determined by eluting authentic samples. The
formation of succinyl CoA and the decrease of CoA
were confirmed. Final concentration: pH 7.8 Tris
buffer 100 mM, succinate 50 mM, GTP 20 mM, CoA
50 mM, histidine methyl ester 100 mM.

Viva Origino 35 (2007) 73 - 81
be discussed in another paper. Figure 5(a) and 6(a)show the decrease of FAD absorption at 448 nm,
and that of fumarase COOHCH=CHCOOH
absorption at 280 nm, respectively.
Figure 5 (b) illustrates the disappearance of a
succinate tenuous spot and the formation of afumarate spot near the origin of elution after thereaction by a TLC experiment. Rf of the fumarate
spot is almost zero, loosing the information fromthis quantity. However, the grey white color of the
spot is completely similar to the fumarate spot in the
Figure 6(b) for fumarate, which endorses theformation of this molecule in the succinate DHreaction. Figure 6(b) is the TLC plate for the
fumarase reaction. The presence of a malate spot
appeared after the reaction in addition of a fumarate
spot near the origin of elution. Malate DH will be
discussed in another letter [3].
Citrate synthase-type activity of histidine:
Acetyl CoA CH3COSCoA and oxalacetate
would
form
citrateCOOHCH2COCOOHCOOHCOCOH(COOH)CH2COOH
andCoA(HSCoA) in the presence of histidine. Figure 7(a)depicts the time sequence of the decrease ofoxalacetate absorption at 290 nm catalyzed byhistidine methyl ester. Nineteen other amino acids(19 aa) showed no activity. Imidazole alone did

not result in the decrease of absorption. Figure 7(b)depicts the result of a TLC experiment: theformation of a citrate spot after the reaction (right)
might be confirmed. In the figure 7(c) for a HPLC
experiment, the formation of CoA and the decreaseof acetyl CoA and oxalacetate after 3 hours wasdemonstrated.

Figure 6(a). (FUM) Time sequence of fumarase
activity of cysteine ethyl ester (CE) observing theabsorbance of fumarate at 280 nm.
Final concentration: pH 7.8 Tris buffer 100 mM,
fumarate 5 mM, cysteine ethyl ester or other aminoacids 100 mM.

Figure 5(a). (SUCD) Time sequence of succinate
dehydrogenase activity of cysteine ethyl ester (CE)observing the absorbance of FAD+ at 448 nm. Finalconcentration: pH 7.8 Tris buffer 100 mM, succinate
10mM, FAD+ 10 mM, cysteine ethyl ester or 19 otheramino acids 100 mM.

Figure 6(b). (FUM) A TLC plate showing theappearance of malate (mal) after the reaction time of
1 day in the presence of cysteine. Final concentration:
pH 7.8 Tris buffer 300 mM, fumarate (fum) 50 mM,
cysteine 100 mM.

Figure 5(b). (SUCD) A TLC plate showing thedisappearance of succinate (suc) and appearance of
fumarate (fum) near origin of development after thereaction time of 1 day in the presence of cysteine.
Final concentration: pH 7.8 Tris buffer 100 mM,
succinate 50 mM, FAD+ 50 mM, cysteine 100 mM.

Figure 7(a). (CITS) Time sequences of citratesynthase activity of histidine methyl ester (Hm)determined by the absorbance of oxalacetate at 290
nm at 250C. Final concentration: pH7.9 Tris buffer
100 mM, oxalacetate 10 mM, acetyl CoA 10 mM, andhistidine methyl ester or 19 other amino acids 100
mM.

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Viva Origino 35 (2007) 73 - 81
3.2. Three histidine enzymes
Here I shall discuss three important histidine
enzymes in addition to amino acid enzymes in the
TCA cycle. Carbamoyl aspartate dehydration is atypical reaction to form nucleic acid ring from a
linear molecule. Together with alanine aminotransformation reaction by histidine in another letter[3] , which is also a typical reaction in amino acidformation, the autotrophic character of genetic
world may be expected. Cleavage of 2-keto-3deoxy

Figure 7(b). (CITS) TLC spots on a cellulose plate forthe citrate synthase activity of histidine methyl ester:(left) the oxalacetate spot was clearly seen by eluting
the reference solution at zero time. Right lane is thatfor the reaction mixture after the reaction time of 1day. Formation of citrate and decrease of oxalacetate
were demonstrated. : oxal for oxalacetate, Hm forhistidine methyl ester, cit for citrate. The final
concentration: pH 7.9 Tris buffer 100mM, oxalacetate
50 mM, acetyl CoA 10 mM, histidine methyl ester
100 mM.

gluconic acid is the most important process in thecentralpathwayofthermophiles(nonphosphorylated Entner-Doudorff pathway), as
shown in a coming paper [4]. However, thismolecule was not obtained commercially.
Consequently, the experiment of cleavage of 2-keto3-deoxy octonic acid here is a good direct example
to certify the possibility above.
Nucleic acid ring formation (Carbamoyl
aspartate dehydratase): Figure 8(a) illustrates theincrease of dihydroorotate absorption at 240 nm in
the presence of histidine. The HPLC diagrams in
Figure 8 (b) of the reaction mixture depicts theformationofdihydroorotonate–NHCONHCH(COOH) CH2CO-, where – means aring connection, peak from the carbamoyl aspartate

Figure 8(a). (CBAD) Time sequence of carbamoyl
aspartate activity of histidine observing theabsorbance of dihydroorotate at 240 nm. Finalconcentration: pH 7.8 Tris buffer 100 mM, carbamoyl
aspartate 25mM, and histidine 50 mM.

Figure 7(c). (CITS) HPLC diagrams of the reaction mixture after 0 and 3 hours for the citrate synthase activity ofhistidine. Formation of CoA after 3 hours was confirmed. The ratio of the integrated absorbance of oxalacetate to thatof acetyl CoA at t=0 is 1/2, while the ratio of oxalacetate to (CoA + acetyl CoA) is1/3.6. Consequently the amount ofoxalacetate was decreased during the time. Final concentration: pH 7.8 Tris buffer 100 mM, oxalacetate 25 mM, acetyl
CoA 25 mM, and histidine 50 mM.

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Viva Origino 35 (2007) 73 - 81

Figure 8(b). (CBAD) HPLC diagrams of the reaction
mixture after 0 and 1.5 hour for the carbamoyl
aspartate dehydratase activity of histidine. The
formation of dihydroorotate was confirmed byobserving the appearance of its peak and decrease of
the carbamoyl aspartate. DO for dihydroorotate andc-asp for carbamoyl aspartate. Final concentration:
the same as in Fig. 8(a).

Figure 9(b). (OCALD). A TLC plate showing theappearance of pyruvate (pyr) and ribose (rib) after thereaction time of 1 day in the presence of histidine(right).
In the left figure, the reference solution at t=0 was
shown, since the white histidine spot hides theoctonate spot. Final concentration: pH 5.9 acetate
buffer 100 mM, 2-keto-3-deoxy octonate 50 mM,
histidine 100 mM.

Figure 9(a) (OCALD). Time sequence of 2-keto-3deoxy octonate aldolase activity of histidine methyl
ester (Hm) using the coupled enzymatic method in thepresence of lactic dehydrogenase to reduce theproduced pyruvate observing the absorbance of
NADH at 340 nm. Final concentration: pH 5.9
acetate buffer 100 mM, 2-keto-3-deoxyoctate 100
mM, NADH 0.12 mM, Rabbit Muscle lactate
dehydrogenase 10 units, and histidine methyl ester or
19 other amino acids 100 mM.

Figure 10(a). (AAD) Time sequence of acetoacetate
decarboxylase activity of histidine methyl ester (Hm)observing the absorbance of acetoacetate at 270 nm.
Final concentration: pH 5.9 phosphate buffer 200
mM, acetoacetate 10 mM, histidine methyl ester 100
mM.

NH2CONHCH(COOH) CH2COOH leaving H2O.
2-keto-3-deoxy octonate aldolase-type activityof histidine: Figure 9(a) shows, by the addition ofhistidine ethyl ester, the decrease of NADH
absorption at 340 nm reacted with the produced
pyruvate from 2-keto-3-deoxy octonate CHOCO
CH2(CHOH)4CH2OH in the presence of lactate DH.
Nineteen other amino acids, the reference had noeffect. Figure 9(b) is the TLC plate showing theappearanceofpyruvateandribosefrom 2-keto-3-deoxy
CHO(CHOH)4CH2OH
octonate in the presence of histidine.
Acetoacetate decarboxylase-type activity ofhistidine: Figure 10 (a) illustrates the decrease ofacetoacetate by measuring the absorption at 270 nm
in the presence of histidine, while 19 other aminoacids and the reference result in no change ofabsorption. Figure 10(b) depicts the formation ofaceton peak from acetoacetate. It also shows thattyrosine had no effect in this reaction.
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4. Discussions on histidine and cysteine enzymes
For the majority of the metabolic reactions
studied above, one amino acid enhances one
reaction rate specifically. Cysteine was found to
mainly participate in the oxidoreductive reactions
and histidine was found to participate in the acidbase reactions. These two amino acids are known to
exhibit activity in the electron and group transfer
reactions, respectively: their side chains containthiol and imidazole, respectively, which are themost electron-mobile group best for catalysis
among those of the twenty amino acids [1].
However, imidazole itself showed no enzymatic
activity. The amino group of histidine is needed.
Formalizationofvariousrate-enhancing
mechanisms of each reaction was not attempted
here. However, it is instructive to discuss thedifference among amino group (in the peptidyl
moiety of amino acids) catalyst, a residue (of aminoacids) catalyst, and an amino acid catalyst. I shallshow in another paper [4] that extraction of waterfrom gluconic acid to form 2-keto-3-deoxy gluconic
acid was catalyzed by various amino acids. In theseViva Origino 35 (2007) 73 - 81
cases, the amino group in the peptidyl moiety of theamino acids is responsible for the reactions. It isalso known that para-nitrophenyl acetic acid ishydrolyzed by histidine. The imidazole group ofhistidine works as a catalyst [1]. In contrast, theamino acid catalyst uses both the amino group(base) and the residue (acid) for catalysis (forinstance, of acid-base type).
One interesting result is that the amino acidrate-enhancers for aconitase and fumarase arecysteine, not histidine which most frequently
appeared in the cases of lyase (dehydratase in thesecases). It is known that aconitase contains sulphur in
its catalytic center [1] similarly to cysteine. Anotherhistidine dehydratase, 2-keto-3-deoxy octonate

aldolase, is essential for cleaving glucose to
pyruvate and glyceraldehyde in the central
metabolic pathway in the ancient bacteria andarchaea. NADH formed in the TCA cycle might
have been oxidized by the nitrate fermentation [7]usingcysteine(corresponding
tonitrateoxidoreductase) and nitrate instead of oxygen andthe electron transfer system in the contemporary
ATPase- membrane system. In the contemporary
biosystem, succinate DH is located in the membrane
and uses FAD  instead of NAD as an oxidant,
and produced FADH is oxidized by the electrontransfer system. Primitively, FAD  may oxidize
succinate to fumarate directly.
5. Origin of Primitive protein Synthetic System
We have already shown that some amino acids
have specific affinity (1 M-1) to their cognate
anticodonic nucleotides in the genetic code table [810]. It was also found [11] that the anticodons in
the contemporary tRNAs could have stronger
specific affinity (200 M -1) to their cognate aminoacids.
In the contemporary biosystem, catalysts areproteins which are formed by the protein synthetic
system containing various complex processes
including amino acids, tRNAs (adaptors),aminoacyl-tRNA synthetases, ribosomes and so on(Figure11( c )).
It is essentially a catalyst
increasing (= protein synthetic) system using RNA.
Since amino acids could possess weak catalytic
activities for some prebiotic reactions, the above
anticodon-cognate amino acid association might beregarded as the oldest protein synthetic system,
although it contained no adaptors yet (Figure 11(b)).
Anticodons were replicable and the “anticodon
world” could be regarded as the smallest genetic
system (in a sense, it forms a smallest RNA world).
The anticodons would be the most primitive“genes” in the meaning that they attract thecatalytic amino acids into the primitive cell (orincrease the numbers of the smallest “enzymes”
there).
For further evolution to elongate the aminoacid to a dipeptide and beyond, we need some

Figure 10(b). (AAD) HPLC diagrams of the above
reaction mixture after 0 and 30 min
for theacetoacetate decarboxylase activity of histidine. The
formation of aceton was observed for histidine, but
not for tyrosine. (Furthermore, addition of Tris buffer
10 µl to the solid mixture of acetoacetate 1 mg andhistidine 1 mg caused degassing of CO2, another goodevidence for the histidine catalyst; (data not shown.).
Final concentration: the same as in Fig.10(a)Figure 11. Evolution of dipeptide formation mechanism in the primitive and Contemporary protein synthetic system.
(a) affinity between an amino acid and its cognate anticodon is the start of the protein synthetic system. (a’) Formationof a dipeptide from two aminoacyl adenylates on 6mer RNA are impossible due to a long distance between the two.
(b) a 6mer RNA (mRNA) with two aminoacyl adenylate on another tRNA can make a dipeptide using their long tails.
The primitive tRNA is also a primitive gene. (c) contemporary system to form a dipeptide drawn quite schematically,neglecting the ribosome and other apparatus. Of course, an anticodon on mRNA sits next to another anticodon.

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Viva Origino 35 (2007) 73 - 81
adaptors - very short tRNAs as discussed by Crick[12]. Since the distance between two amino acid ona 6mer RNA is too great from each other to make abond using some energy-supplying molecule(Figure 11(a’)), Crick assumed the presence of an
adaptor with anticodonic trinucleotides at its 5’ end
and a tail having an aminoacylated nucleotide at its
3’ end (Figure 11(b)). The shortest primitive tRNAof 25 mers with an anticodon at its 5’end and a stem
plus the discriminator and CCA trinucleotides at its
3’end was shown to be aminoacylated using
aminoacyladenylates and a varylaspartate as thecatalyst [13]. The number of the 7 base pairs in thestem could be reduced to 2 (Shimizu, unpublishedresult). Hopefully, the final goal of this type ofapproach may be to find a system of 6 mer tRNAs(as a template for the dipeptide, so it could be theprimitive gene) and dipeptide catalysts foraminoacylate formation and elongation (Figure11(b)) . Figure 11 illustrates the above discussion.
6. Relationships of amino acid catalyst to thedipeptide catalysts
I have demonstrated that some dipeptides
could function as catalysts in various biochemical
reactions: as already discussed, valylaspatate (orvarylglutamate) behaved as an aminoacyltransferase[13]. In this relationship, I found thatglutamic acid has transferase activity in this paper.
Similarly, histidine frequently appeared in the
dipeptide catalysts, such as alanylhistidine [14],serylhistidine and glutamylhistidine[15]. The
catalytic activity of amino acids had more increasedin the dipeptide catalysts. More precise specificity
to substrates and higher activity of biocatalysts
would have sometimes been sophisticated by theincrease in the numbers of amino acids in the
catalysts (peptides or proteins) through biochemical
evolution.
7. Three concentration mechanisms of amino
acids
Amino acid and anticodon rate-enhancers are
all weak. Consequently, there should be someconcentration mechanisms to increase theconcentrations of these compounds in the primitive
oceans, if these compounds were correlated with thegeneration of the life there. Three mechanisms forconcentrations in a primitive cell on the primordial
earth could be considered:(1) Evaporation by heat: About 3.9 billion
years ago, the earth began to cool due to the ceaseof the infall of planetesimals but locally was still
very hot. This was a unique time for generation oflife on this planet. From carbon isotope data,
photosynthesis would have worked at this time[16].
The concentration of the amino acids in a pool at
the seashore near volcanoes would have reached ahigh level.
(2) large fluctuation.: Concentration of aminoacids in one of the primitive cells might haveincreased to 10-100 mM level by large fluctuation(see the random drift in Kimura’s neutral theory[17] for the evolution of species at a molecularlevel). Dyson also stressed the importance of
rapider random drift against slow selection [18]. It
is noteworthy that this discussion could apply to thegeneration of life in the hydrothermal vents at the- 80 -

bottom of oceans, where employment of theconcentration mechanism by evaporation would be
difficult.
(3) Specific association: If some anticodonic
RNAs (about 1.5 nm size) were contained in theprimitive cell, the RNA might have formed acomplex with an amino acid (about 0.5 nm size)
entering the cell from outside. This complex, whichhave a much larger size than a single amino acid,would have remained in the cell for a longer timethan an amino acid would, contributing to anincrease in the amino acid concentration.
8. Conclusion
Amino acid-metabolites interactions in thecase of the TCA cycle are studied using ultravioletvisible spectroscopy. These are confirmed by cold
thin layer chromatography
and high performanceliquid chromatography.
The anticodon-codon relationship is the trunkof the concept for inheritance. (In contrast, there isno specific relationship between amino acids.
Metabolite-metabolite relationships are mereorganic chemistry.)
The determination of the
DNA structure with a simple symmetry resulted in
an intuitive molecular understanding of inheritance.
On the other hand, metabolism has no simple
symmetry in it and contains so many reactions. The
information here and that on the central pathway inanother paper [4] may be useful for understandingof primitive metabolism at the small molecularlevel.
In the genetic code world, the amino acidworld and anticodon world are coupled inseparably,
supporting both inheritance and metabolism [3].
The anticodon world is the minimal RNA world.
The usual RNA polymer (~100mers) world
suggested by SELEX faced a great difficulty
recently by the finding of high primitive ocean
temperature (~80 oC) due to a good correlation
between two mutually independent O (from sea
water) and Si (from outside silica) isotope data forold cherts [3,19]. It is noteworthy that amino acids
and anticodon could be formed at high temperaturesuch as that of hydrothermal vent.
Acknowledgment
The author thanks to Prof. K.Watanabe ofFaculty of Technology, the university of Tokyo(now at Biological Information Research Center,
National Institute of Advanced Industrial Scienceand Technology). He also appreciates Prof.
H.Himeno of Faculty of Science, Hirosaki
University for his useful discussions.
References
1. Voet, D. and Voet, J. D. Biochemistry, second
edition, John Wiley and Sons Inc., New York,1995.
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