Biological Role of Inorganic Pyrophosphate

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Library of Congress Cataloging-in-Publication Data

Heinonen, JukkaK., 1937-

Biological role of inorganic pyrophosphate / by Jukka K. Heinonen.
p.cm.
Includes bibliographical references and index.
ISBN 978-1-4613-5551-9 ISBN 978-1-4615-1433-6 (eBook)
DOI 10.1007/978-1-4615-1433-6
1. Pyrophosphates--Physiological effect. 2. Pyrophosphates--Metabolism. 1. Title.

QP535.Pl H4542001
572'.553--dc21

2001038127

Copyright © 2001 Springer Science+Business Media New York. Second Printing 2003.
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CONTENTS
PREFACE .............................................................................. vii

ACKNOWLEDGEMENTS ........................................................ ix

ABBREVIATIONS .................................................................. xi

1. BIOLOGICAL PRODUCTION OF PPj .................................... ..

1.1. PP j producing reactions ...................................................... ..
1.1.1. Number and types of the reactions.......................................... 1
1.1.2. Why is PPj produced in biosynthetic reactions.. . ... ..... . ... ... ... ... ... 10
1.1.3. PPj synthesis using chemical gradients ... . . . .. . . . . . . . . . . . . . . . . .. . . . . . .. . . . .. 14
1.2. Rate and amount of PP j production in biochemical reactions... ... ... 23

2. DETERMINATION OF PPj CONCENTRATION IN LIVING

SYSTEMS .............................................................................. 29
2.1. Sampling and extraction ..................................................... . 29
2.2. Assays ............................................................................ .. 34
2.2.1. Radiochemical determinations............................................... 34
2.2.2. Colorimetric PPj assays....................................................... 36
2.2.3. Enzymatic methods............................................................ 37
2.2.4. Physical methods.. . ... .. . . .. ... ... .. . ... ... ... ..... . . .. .. . . .. ... ... ... .. . ...... 41
2.2.5. Calculation of PPj concentration from the equilibrium constant of a
biochemical reaction... ... . .. ... .. . . .. ... .. . ... ... .. . . .. .. . ... ... ... ... ... ... . .. ... ... 42
2.3. Conclusions... ... . .. ... ... .. . .. . .. . ... ... . .. ... . .. ... . .. ... ... ... ... ... ... ... ..... 43

3. PPjCONCENTRATlON IN BIOLOGICAL MATERIAL ............... 45

3.1. Bacteria ........................................................................... 45
3.2. Lower eukaryotes ......... . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 48
3.3. Plants... . .. ... ... ..... . ... ... ... ... ... .. . ... ... . .. ... .. . ... . .. ... . .. ... ...... . .. ... 53
3.4. Animals ........................................................................... 60
3.5. Humans ............................ , ...... , .. . . .. . .. ... . .. . .. ... ... ... ... ... ... ... 65
3.5.1.Blood ............................................................................ 65
3.5.2. Urine............................................................................. 69
3.5.3. Synovial fluid.................................................................. 73
3.5.4. Saliva............................................................................ 73
3.5.5. Bone, tooth, and cartilage ..................................................... 74
3.5.6. Cultured human cells .......................................................... 74

4. PP j AS A BIOCHEMICAL ENERGY SOURCE .......................... 77

4.1. General considerations...... ... ...... ... ... ...... ...... ... .. .. .. .. .. .. .. ....... 77
4.2. Bacteria ............... . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.3. Protists ............................................................................ 89
4.4. Plants... .. . ... ... .. . ... ... .. . ... ... .. .... ... . .. .. . ... ... .. . . .. ... . ..... ... .. . ... . .. 93
4.4.1. PPj-dependent enzymes in plants...... ...... .... .. .. .. .. ...... ...... ......... 95
vi
4.4.2 Role ofUDPG-pyrophosphorylase .......................................... 101
4.4.3. Role ofH+ -translocating PPase ............................................... 103
4.4.4. Role of PP j -dependent phosphofructokinase ....................... , . . .... 105
4.4.5 Conclusions..................................................................... III
4.5. Animals ........................................................................... 112
4.6. Other eukaryotes .................. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . .. 115
4.7. PP j as an energy source of primitive life ............... . . . . . . . . . . . . . . . . . . ... 117

5. REGULATORY ROLES OF PP j ••••••••••••••• '" •••••••••••••••••••••••••••• 123

5.1. PP j as a regulator of enzyme activity ........................................ 123
5.1.1. Binding of metal ions......................................................... 123
5.1.2. Product inhibition .............................................................. 125
5.1.3. Other cases .................................. , ................................... 134
5.1.4. PP j as an activator of enzymes ..... , .......................................... 141
5.1.5. PP j and adenine nucleotide translocase ................... , .................. 144
5.2. Regulation of nucleic acid and protein synthesis ......................... 148
5.2.1. Fidelity of the syntheses ...................................................... 148
5.2.2. PP j in proofreading ............................................................ 149
5.3. PP j and calcification... ... . .. ... ... ... .. . ... ... ... .. . .. . . . . . .. ... ... .. . .. . .. . ... 159
5.3.1. Bone formation and resorption ..................... '" ....................... 159
5.3.2. Tooth ............................................................................. 166
5.3.3. Urolithiasis .............................. '" ..................................... 168
5.3.4. Pathologic c;alcification of soft tissues. .. .. . .. . . .. .. . .. . ... .. . ... . . . .. . . .... 171
5.4. Effect of extracellular PP j • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • ••. 173
5.4.1. PP j and cell proliferation ................................................... '" 173
5.4.2. CPPD crystal deposition disease ............................................. 177
5.5. PP j and iron metabolism ........... , .......................................... 183

INDEX ................................................................................. 189

REFERENCES .......... , ........................... , ......................... ,. ... 197

 vii
PREFACE

This book "grew" naturally during my career as a researcher in biochemistry. For

more than three decades I have been interested in the role of inorganic pyrophosphate
(PPi) and that of the enzyme hydrolysing it (pPase). My first paper on this topic
appeared in 1970. My fascination on PPi has two reasons. First, being a kind of
maverick by nature, I prefer to study unfashionable topics, and most biochemists have
thought (and still do, as biochemical textbooks attest) that PPi is only a waste product
that must be destroyed immediately to facilitate the biosyntheses of macromolecules.
Second, PPi has (or at least has been proposed to have) a role in many biological
processes such as cellular energy metabolism, regulation of enzyme activity,
regulation of the fidelity of the syntheses of biological macromolecules, bone
formation and pathological calcification, the etiology of a pathologic condition of
joints, and the emergence of life on Earth. Therefore, in satisfying my curiosity on the
biological role of PPi, I have been able to get acquainted with many widely different
facets of life without being suffocated by the excessive amount of data that is available
on more fashionable areas of biochemistry.
This book is an attempt to review and critically evaluate all the scientific literature
on the biological role of PPj published from the 1940s to the end of the year 1999.
The data scattered in biochemical, botanical, microbiological, and medical literature
has been collected and presented in a concise form, which should be understandable to
an average scientist graduated in biochemistry or related subjects. To facilitate this,
short general introductions to subtopics with references to fresh reviews have been
presented, when considered necessary. Experimental methods are described in such
details that the original papers are not required to understand, what and how has been
done. When possible the data is critically evaluated as to its reliability and biological
significance, discrepancies are pointed out, and solutions to them, as well as needs of
further research are proposed. As far as possible the original papers in international
journals are referred to, while congress abstracts, and PhD dissertations are avoided,
because they are often not generally available (as I have clearly found out). Naturally
pertinent reviews on any subtopic of this book are also included.
In the five chapters of this book I review and discuss the Ppj-producing
biochemical reactions, the determination and concentration of PPj in biological
material, PPj as a biochemical energy source and its regulatory roles.
In the first chapter all (almost 200) known biochemical reactions that produce PPj
are presented in the tabulated form. The reactions are classified according to the type
of the reaction and the role of each reaction in living cells. The EC. numbers of the
catalysing enzymes are also included, when available. Further in this chapter I apply
quantitative bioenergetics to look for an explanation, why some biochemical reactions
are energised by the hydrolysis of nucleoside triphosphate into PPi and
monophosphate, while others use the hydrolysis to orthophosphate and nucleoside
diphosphate. The third subchapter is devoted to the direct formation of PPj from
orthophosphate using the energy of biological concentration gradients. Finally
published biochemical data is used to estimate the rate and amount of PPj production
in some living systems.
In the second chapter all published methods used in biological PPj assays are
described and critically evaluated and also some new possibilities are proposed.
viii
The available data on PPi concentration in living material is compiled and
critically discussed in the third chapter. My conclusion is that contrary to common
notion, PPi exists throughout the living world in rather high concentrations (10-5_10-3
molar).
Finally the main topic of this book, the biological role of PPi, is thoroughly
reviewed and discussed in the last two chapters.
Many biochemical reactions that use PPi instead of ATP as a biochemical energy
source have been described in the scientific literature in the last four decades. Such
reactions are especially important in some species of bacteria and protists and most of
all in common plants. All these reactions are presented in the chapter four and their
significance is evaluated from the bioenergetic and regulatory points of view. In the
first subchapter PPi is compared with ATP as a biochemical energy source, while in
the last one the possibility that PPi might be an evolutionary precedent of ATP is
considered.
A large body of data has been published showing that PPi can affect many
biochemical and physiological reactions and this data is presented and critically
discussed in the last chapter of this book. PPi has been reported to influence the
activities of many enzymes, although the biological significance of these observations
is dubious in most cases. It may also affect the accuracy of syntheses of biological
macromolecules, but the final verdict is not yet available. In the last three subchapters
the medical aspects of PPi in living systems are described. Many results have been
published suggesting that PPi has a role in bone formation and some papers have
appeared, where it has been proposed to affect cell proliferation and iron transport
into cellular compartments. Pathologic aspects of PPi metabolism include bone
diseases, calcification of soft tissues, and formation of urinary stones, although the
evidence for the role ofPPi is not conclusive in these cases. PPj is certainly involved in
pseudogout, where crystals of calcium pyrophosphate dihydrate accumulate in joints,
mostly knees. This ailment is rather common among aged people and has therefore
been the object of many studies from the 1960s to the present time.
I dare to believe that his book is a unique and invaluable source of references and
digested data to all professional scientists, who study or are planning to start studies
on the the role of PPj in living systems. These scientists are specialists in many
different branches of science (biochemistry, microbiology, bioenergetics, plant
physiology, parasitology, evolution, orthopedics, rheumatology etc.) and publish
mostly in different journals, but this book sums up all the knowledge in one book. My
wish is that it aids scientist to cross the boundaries between different disciplines. This
book could also help any scientist to enlargen his or her scientific literacy by giving an
overview on this interesting and multifacetted, but somewhat neglected topic.
 ACKNOWLEDGEMENTS

My sincere thanks belong to three persons, who gave me invaluable practical support,
while I wrote this book: The librarian Into Wester saved me many hours and
countless steps by ordering and carrying to me the papers and books that were not
available in the departmental library. The reference list was prepared and checked by
Mr Toni Sivula, and Dr Pekka Rappu served patiently as my computer expert. I am
grateful also to all members of our PPase reserch group for the stimulating and
congenial atmosphere, where this book was born. Especially I want to thank Dr Reijo
Lahti and Dr Anu Salminen, who have critically read parts of the manuscript.
 ABBREVIATIONS
ACMA = 9-amino-6-chloro-2-methoxyacridine
ADP adenosine diphosphate
AMP adenosine monophosphate
ANT adenine nucleotide translocase
APS adenylyl sulfate
ATP adenosine triphosphate
ATP-PFK =ATP-dependent phosphofructokinase
AZT = azidothymidine
CAM = crassulacean acid metabolism
CaMV = cauliflower mosaic virus
CCCP = carbonyl cyanide m-chlorophenylhydrazone
CoA = coenzyme A
cNMP cyclic mononucleotide
CPPD = calcium pyrophosphate dihydrate
dNTP = any deoxyribonucleotide triphosphate
EDTA = ethylenediamine tetraacetate
EGF = epidermal growth factor
F2,6BP = fructose-2,6-bisphosphate
FBP = fructose-1,6-bisphosphate
FCCP = carbonyl cyanide p-trifluoromethoxyphenylhydrazone
g fw = grams of fresh weight
HAP = hydroxyapatite
NAD+ = nicotinamide adenine dinucleotide oxidized form
NADH = nicotinamide adenine dinucleotide reduced form
NADP+ = nicotinamide adenine dinucleotide phosphate
NMN = nicotinamide mononucleotide
NMP = any ribonucleoside monophosphate
NTPPPH = nucleoside triphosphate pyrophosphohydrolase
NTP pyrophosphatase
P 3 = tripolyphosphate
PCA = perchloric acid
PDGF = platelet derived growth factor
PEr = polyethylene imine
PEP = phosphoenolpyruvate
PFP = pyrophosphate:fructose-6-phosphate 1-
phosphotransferase = PPi-dependent
phosphofructokinase
Pi inorganic orthophosphate
PPase = inorganic pyrophosphatase
PP i = inorganic pyrophosphate
PPi-PFK = PPi-dependent phosphofructokinase PFP
PRPP = 5'-phosphoribosyl-l'-diphosphate
PRTase = phosphoribosyltransferase
RME receptor mediated endocytosis
SAM S-adenosylmethionine
TCA trichloroacetic acid
TGF transforming growth factor
 1. BIOLOGICAL PRODUCTION OF PP,

1.1. PPi producing reactions

l.l.1 Number and types of the reactions

Inorganic pyrophosphate (PPi) was discovered already in the nineteenth century.

As its name implies, it is synthesized by heating sodium or potassium salts of
orthophosphate (see van Wazer 1958). Formation of PPi in a biological system was
reported in 1941 by Cori, who found that it accumulated in rat liver extract incubated
aerobically in the presence of succinate and fructose (referred in Cori et al. 1951).
The first biological reaction, where PPi was formed was described by Kornberg in
1948. He found that yeast cell extract catalyzed formation of ATP and NMN in the
presence of NAD+ and PPi. He proposed the name pyrophosphorolysis for this
reaction in analogy with the already known phosphorolysis. The reaction was readily
reversible and thus PPi and NAD+ were the products, if ATP and NMN were used as
substrates. In the 1950s many similar reactions were observed, and in 1957 Kornberg
proposed in a review article that in vivo pyrophosphorylases mostly act in the
direction of PPi formation serving the biosynthesis of stable biochemical compounds.
Coupling to hydrolysis of PPi by inorganic pyrophosphatase makes these reactions
practically irreversible. This hypothesis, which is now generally accepted, was stated
more firmly by him in 1962.
In 1961 Imsande and Handler described 22 different pyrophosphorylases, which
they divided into 5 groups according to the type of reaction catalysed by them:
a. The reaction of a nucleoside triphosphate (NTP) with a phosphate ester of a
primary alcohol.
b. The reaction ofNTP with a glycol phosphate.
c. The reaction of 5-phosphoribosyl-pyrophosphate (PRPP) with a compound
containing a quarternary nitrogen.
d. Pyrophosphorylation of DNA
e. Activation of precursors of biosynthesis at the expense of NTPs.
They also argued that pyrophosphorylase is a misnomer for these enzymes, because in
vivo these reactions go to the direction ofPPi formation.
In 1962 Kornberg listed the then known biosynthetic reactions that produce PPj
and this table already contained most such reactions that have been described up to
now. I have collected into Table 1.1 all PPj producing biochemical reactions that I
could fmd in the end of 1999. Because the review articles of Imsande and Handler
(1961) and of Kornberg (1962) are already almost 40 years old and may not be
available everywhere, I have included all the reactions given by these authors. The
references in the Table l.1 indicate the paper, where, as well as I was able to
ascertain, it was for the first time clearly stated that PPj is formed in the reaction
shown. Also the references given by Imsande and Handler (1961) and by Kornberg
(1962) have been checked and in some cases changed on this basis.
2

Table 1 1 PPi Releasing Biochemjcal reactjons

1. NTP + X + Y = X-Y + AMP + PPi

1.1. Synthesis ofaminoacyl-tRNA
ATP + an amino acid + tRNA = aminoacyl-tRNA + AMP + PPi.
6.1.1.1-7; 6.l.1.9-12 ; 6.1.1.14-22. Hoagland et al. 1958.

1.2. Cell wall synthesis in some bacteria

ATP + D-alanine + poly(ribitolphosphate) = O-D-alanyl-poly(ribitolphosphate) + AMP +
PPi.
6.1.1.13. Baddiley and Neuhaus 1959.

1.3. Activation of carboxylic acids

ATP + R-COO- + Coenzyme A = R-CO-Coenzym.e A + AMP + PPi.
6.2.1.1-3; 6.2.1.7-8; 6.2.1.11-12; 6.2.l.14-17; 6.2.1.23-33. Chou and Lipmann 1952.

1.4. Protein degradation in vivo

ATP + ubiquitin + protein-lysine = protein-N-ubiquityllysine + AMP + PPi.
6.3.2.19; 6.3.2.21. Chiechanover et al. 1981.

1.5. Nonribosomal peptide synthesis

a. Synthesis of antibiotic peptides in bacteria
nATP + namino acids = peptide + nAMP + nPPi.
No EC number. Gewers et al. 1968.
b. ATP + L-histidine + 6-alanine = carnosine + AMP + PPi.
6.3 .2.11. Kalyankar and Meister 1959.

c. Synthesis of kyotorphin in brain

ATP + L-arginine + L-tyrosine = L-tyrosyl-L-arginine + AMP + PPi.
6.3.2.24. Veda et a/1987.

1. 6. SyntheSiS ofa fJ-Iactam ring

N2-(2-carboxyethyl)arginine + ATP = deoxyguanidinoproclavaminate + AMP + PPi.
No EC number. P-Iactam synthetase. Bachmann et al. 1998.

1.7. Amide synthesis

ATP + an amino acid + NH/ = the corresponding amide + AMP + PPi or
ATP + an amino acid + L-glutamine = the corresponding amide + L-glutamate + AMP +
PPi.
6.3.1.1 ; 6.3.1.7 ; 6.3.5.4. Ravel et al. 1962.
 3

I.B. Arginine and urea synthesis

ATP + L-citrulline + L-aspartate = L-argininosuccinate + AMP + PPi.
6.3.4.5. Petrac and Rattner 1958.

1.9. Synthesis oj coenzymes

a. biosynthesis ofNAD.
ATP + deamidoNAD+ + ammonia = NAD+ + AMP + PPi or
ATP + deamidoNAD+ + L-glutamine = NAD+ + L-glutamate + AMP + PPi. 6.3.1.5 ;
6.3.5.1. Preiss and Handler 1958.
b. biosynthesis of coenzymeA.
ATP + (R)-pantoate + B-alanine = (R)-pantothenate + AMP + PPi.
6.3.2.1. Maas and Novelli 1953.
CTP + 4-phosphopantothenate + L-cysteine = 4-phosphopantothenoylcysteine + CMP +
PPi.
6.3.2.5. Jackowski 1996.

1.10. Guanylate synthesis

ATP + XMP + ammonia = GMP + AMP + PPi or
ATP + XMP + L-glutamine = GMP + L-glutamate + AMP + PPi.
6.3.4.1. ; 6.3.5.2. Lagerqvist 1957.

1.11. Covalent modification oJproteins with carboxylic acids

a. ATP + biotin + apoenzyme-lysine = apoenzyme-N-biotinyllysine (holoenzyme) +
AMP+PPi.
6.3.4.9 - 11 ; 6.3.4.l5. Lane et al. 1964.
b. ATP + lipoate + apoenzyme-lysine = apoenzyme-N-lipoyllysine (holoenzyme) + AMP
+pp,
No EC number. Reed et al. 1958.
c. ATP + carboxylate + HS-enzyme = carboxyl-S-enzyme + AMP + PPi.
6.2.1.19-20; 6.2.1.22. Schmellen and Eggerer 1974.

1.12. Ligation oJRNA and DNA

a. ATP + (NMP)n-3'-OH + P-5·-(NMP)m = (NMP)n+m + AMP + PP,.
6.5.1.1 ; 6.5.1.3. Weiss and Richardson 1967.
b. ATP + RNA = RNA-terminal-2'3'-cyclic phosphate + AMP + PPi.
6.5.1.4. Filipovicz et al. 1985.

1.13. SynthesiS oJphosphoenolpyruvate in C4 photosynthesis

ATP + pyruvate + ortophosphate = PEP + AMP + PPi.
2.7.9.1. Hatch and Slack 1968.

2. ATP+ X=Y + AMP + PPi

4

2.1. Reduction 0/a carboxyl group

a. ATP + aryl-COO- + NADPH = aryl-CHO + NADP+ + AMP + PPi.
1.2.1.30. Gross 1971.
b. biosynthesis oflysine in fungi
ATP + L-a-aminoadipate + NADPH = L-a-aminoadipate-o-semialdehyde +
NADP+ + AMP + PPi.
No EC number. Sagisaka and Shimura 1962.

2.2. Light production by firefly luciferase

ATP + luciferin + 02 = oxidized luciferin + C02 + H2O + light + AMP + PPi.
1.13.12.7. McElroy and Green 1956.

2.3. Racemization o/phenylalanine

2.3. ATP + L-phenylalanine + H2O = D-phenylalanine + AMP + PPi.
5.1.1.11. Yamada and Kurahashi 1969.

3. NTP + X = NMP-X + PPi

3.1. Activation o/sugars

NTP + sugarphosphate = NDP-sugar + PPi.
2.7.7.9-11 ; 2.7.7.13 ; 2.7.7.23-24 ; 2.7.7.27-30 ; 2.7.7.32-34.
Munch-Petersen et al. 1953.
No EC number. NTP-p-glycero-D-mannoheptose-l-phosphate nucleotidyltransferase.
Eidels and Osborn 1971.
CTP-myoinositol-l-phosphate cytidylyltransferase. Chen et al. 1998.
CTP-2-C-methylerythritol-4-phosphate cytidylyltransferase. Rohdich et al. 1999

3.2. Activation o/various other compounds for biosyntheses o/phospholipids,

antibiotics etc.
CTP + cholinephosphate = CDP-choline + PPi.
2.7.7.14-15 ; 2.7.7.38-41 ; 2.7.7.43-44 ; 2.7.7.46-47 ; 2.7.7.55 ; 2.7.7.57-58.
Kennedy and Weiss 1956.

3.3. Sulfate activation

ATP + sulfate = APS + PP,.
2.7.7.4. Hilz and Lipmann 1955.

3.4. Synthesis 0/coenzymes

a.ATP + nicotinamide ribonucleotide = NAD+ + PP,.
2.7.7.1 ; 2.7.7.18. Kornberg 1948.
b. ATP+FMN=FAD+PP,.
2.7.7.2. Schrerer and Kornberg 1950.
c. ATP + pantetheine-4' -phosphate = dephospho-coenzyme A + PP,.
2.7.7.3. Hoagland and Novelli 1954.
d. GTP + adenosylcobinamide phosphate = adenosylcobinamide-GDP + PPi (aerobic
 5

synthesis of vitamin BI2).

No EC number. Adenosylcobinamide phoshate guanylyltransferase. Barchielli et al. 1960.
e. GTP + 5-hydroxy-6-methylbenzimidazolylcobamide = cobinamide-GDP + a-ribazole +
PPi (anaerobic synthesis of vitamin B12).
No EC number. Schulze et al. 1998.

3.5. Nucleic acid synthesis

NTP + (NMP)n + template = (NMP)n+ I + template + PPi.
2.7.7.6-7; 2.7.7.48-49. Bessman et al. 1957.

3.6. Modification of nucleic acids

a. Polyadenylation of mRNA
nATP + RNA-3'-OH (no template) = RNA-3'-(AMP)n + nPPi.
2,7.7.19. Edmonds and Abrams 1960
b. Template-independent addition ofnucleotides to DNA
NTP + DNA-3'-OH = DNA-3'-NMP + PPi,
2.7.7.31. Krakowet al. 1961.
c. Capping of mRNA
GTP + (5')ppPur-mRNA = GpppPur-mRNA + PPi.
2.7.7.50. Martin and Moss 1975.
d. RNA catalysed self-capping
GDP + 5 'pppRNA = GpppRNA + PPi.
No EC number. Artificial ribozyme. Huang and Yaros 1997
d. Formation of the acceptor end of tRNA
NTP + tRNA-3'-OH = tRNA-3'-NMP + PP,.
2.7.7.21 ; 2.7.7.25 ; 2.7.7.52. Hecht et al. 1958.

3.7. Oligonucleotide synthesis

a. (n+I)ATP = pppA-2'-(P-5'-A)n +nPPi.
No EC number. Justesen et al. 1980.
b. 2GTP = bis-guanosyltetraphosphate + PPi.
2.7.7.45. Warner and Huang 1974.

3.8. Regulation ofglutamine synthetase

a. nATP + glutamine synthetase (more active) = glutamine synthetase (AMP)n
(less active) + nPPi.
2.7.7.42. Kingdon et al. 1967.
b. UTP + protein 11 = protein II(UMP) + PPi.
2.7.7.59. Mangum et al. 1973.

3.9. Modification of coenzymes

ATP + FactoC42o = FactoC39o (Factor42o-AMP) + PPi.
No EC number. Factor-390 synthetase. Kengen et al. 1989,

3.10. Activation ofL-malate for polymalate synthesis

ATP + L-malate = ~-L-malyl-AMP + PPi.
No EC number. ~-l-malyl-AMP ligase. Willibald et al. 1999.
6

4. NTP +X=N-X+PP, + Pi

4.1. Synthesis ofS-adenosylmethionine and adenosylcobalamin

ATP + L-methionine + H20 = SAM + PPi + Pi.
2.5.1.6; 2.5.1.17. Mudd and Cantoni 1958.

5. NTP = NMP + PPi

5.1. Synthesis ofcyclic nucleotides

NTP = cNMP + PPi.
4.6.1.1-2; 4.6.1.6. RaIl and Sutherland 1962.

5.2. Hydrolysis ofNIP

NTP + H20 = NMP + PPi.
3.6.1.8; 3.6.1.12 ; 3.6.1.19; 3.6.1.23. Heppel and Rilmoe 1953.

5.3. Synthesis ofhistidine

N 5-(Phosphoribosyl)ATP + iliO = N 5-(phosphoribosyl)AMP + PPi.
3.6.1.31. Smith and Ames 1965.

5.4. Synthesis offolic acid

ili-neopterin triphosphate + H2O = H2-neopterin monophosphate + PPi.
No EC number. Dihydroneopterin triphosphate pyrophosphohydrolase.
Suzuki and Brown 1974.

5.5. Synthesis ofpuromycin

3'-amino-3'-deoxyATP = 3'-amino-3'-deoxyAMP.
No EC number. Pur7 protein of Streptomyces alboniger. Espinosa et al. 1999.

6. Pbospboribosylpyropbospbate + X = X-ribose--5'-pbospbate + PPi

6.1. Nucleotide synthesis de novo

a. pyrimidine nucleotides
PRPP + orotate = OMP + PPi.
2.4.2.10. Liebetman et al. 1954.
b. purine nucleotides
PRPP + L-glutamine + iliO = 5-phosphoribosylamine +L-glutamate + PPi.
2.4.2.14. Goldwait et al. 1955.

6.2. Nucleotide synthesis, salvage reaction

PRPP + N = NMP + PPi.
2.4.2.7-9; 2.4.7.22. Kornberg et al. 1955.

6.3. Amino acid biosynthesis

a. histidine; PRPP + ATP = (5-phosphoribosyl)ATP + PPi.
2.4.2.17. Amesetal. 1961.