GOMBE STATE UNIVERSITY

FACULTY OF SCIENCES

DEPARTMENT OF BIOCHEMISTRY

COURSE:

ASSIGNMENT SUBMITTED TO THE COURSE LECTURER

BY

UMAR HASSAN

PG19/PGD/BCHM/1004

SYNTHESIS OF PURINE AND PYRIMIDINE NUCLEOTIDES,

REGULATIONS AND DISORDERS

AUGUST 2021
INTRODUCTION

Biosynthesis is a multi-step, enzyme-catalyzed process where substrates are converted into more
complex products in living organisms. In biosynthesis, simple compounds are modified,
converted into other compounds, or joined together to form macromolecules. This process often
consists of metabolic pathways. The purines are built upon a pre-existing ribose 5- phosphate.
Liver is the major site for purine nucleotide synthesis. Erythrocytes, polymorph nuclear
leukocytes & brain cannot produce purines.

There are two pathways for the synthesis of nucleotides:

1. De-novo synthesis: Biochemical pathway where nucleotides are synthesized from

new simple precursor molecules

2. Salvage pathway: Used to recover bases and nucleotides formed during the degradation of
RNA and DNA

Composition of Nucleotides

Nucleotides, comprising a deoxyribose (sugar), a phosphate group, and a nitrogen base,

constitute the deoxyribonucleic acid (DNA) backbone.

There are four nitrogen bases, and therefore, four types of nucleotides. These four nitrogen-
containing bases are as follows:

I. Adenine

II. Guanine

III. Cytosine

IV. Thymine

The resulting nucleotide is named accordingly. For example, if the base is adenosine, the
nucleotide is known as deoxyadenosine-5’-monophosphate. The nucleotides are abbreviated with
the initials of their base, i.e. A, G, C, and T.
Nitrogen bases are grouped into two categories; adenine and guanine constitute the purine
category, whereas cytosine and thymine form the pyrimidine class.

SYNTHESIS OF PURINE AND PYRIMIDINE NUCLEOTIDES

DENOVO SYNTHESIS OF PYRIMIDINE NUCLEOTIDES

The intermediate product of pyrimidine synthesis is initially a ribonucleotide. During this

process, the ribose is reduced to 2’-deoxyribose, which can be incorporated into the DNA.

The biosynthesis of pyrimidine nucleotides occurs over multiple steps involving different
enzymes. The pyrimidine ring is synthesized first and the ribose sugar is subsequently added to
it.

In the first step of pyrimidine synthesis, the carbamoyl phosphate and aspartate react to produce
carbamoyl aspartate along with the release of a phosphate moiety. This reaction is catalyzed by
aspartate transcarbamoylase.

Next, dihydroorotic acid is oxidized to orotic acid in the presence of orotic acid dehydrogenase.
This enzyme uses NAD+ as a coenzyme yielding NADH and H+ as the byproducts.
Orotic acid reacts with phosphoribosyl pyrophosphate (PRPP) to form orotidine-5-phosphate
with the release of pyrophosphate. This reaction is mediated by orotate
phosphoribosyltransferase.

Orotidine-5-phosphate undergoes decarboxylation to form uridine-5-phosphate (UMP) in the

presence of orotidine-5-phosphate decarboxylase.

Uridine-5-phosphate constitutes the building block of the subsequent reactions. First, UMP is
phosphorylated to form UDP, which can be further phosphorylated to UTP. The phosphate group
required for this reaction is obtained from the conversion of ATP to ADP.

Following an ATP- and glutamine-dependent reaction, UTP is converted to cytidine triphosphate

(CTP) in the presence of CTP-synthetase.

On the other hand, uridine-5-phosphate can be reduced to d-UMP by the action of d-TMP-
+
synthetase (thymidylate-synthase). This reduction is NADPH H+-mediated. The subsequent
reaction catalyzed by d-TMP-synthetase is the methylation of d-UMP to d-TMP. The methyl
group that is required for this conversion is obtained from N 5, N10-methylene tetrahydrofolate,
which in turn is converted to dihydrofolate.

Ribonucleotide reductase catalyzes this reaction in the presence of thioredoxin as a cofactor.

Thioredoxin, in turn, contains two SH groups, which are converted to the disulfide form after
reduction. Thioredoxin, in its disulfide form, is reconverted to its original form through NADP +-
dependent thioredoxin reductase.

STEPS INVOLVES IN THE SYNTHESIS OF PURINE NUCLEOTIDES

Ribose-5-phosphate, of carbohydrate metabolism is the starting material for purine nucleotide

synthesis.

 It reacts with ATP to form phosphoribosyl pyrophosphate (PRPP). • Glutamine transfers its
amide nitrogen to PRPP to replace pyrophosphate & phosphoribosylamine. Catalysed
produce 5- by PRPP glutamyl amidotransferase.

 This reaction is the committed. • Phosphoribosylamine reacts with glycine in the presence of
ATP to form glycinamide ribosyl 5- phosphate or glycinamide ribotide (GAR).Catalyzed by
synthetase.

 N10-Formyl tetrahydrofolate donates the formyl group & the product formed is
formylglycinamide ribosyl 5-phosphate. Catalyzed by formyltransferase.

 Glutamine transfers the second amido amino group to produce formylglycinamidine ribosyl
5- phosphate. Catalyzed by synthetase.

 The imidazole ring of the purine is closed in an ATP dependent reaction to yield 5-
aminoimidazole ribosyl 5-phosphate. Catalyzed by synthetase

 Incorporation of CO2 (carboxylation) occurs to yield aminoimidazole carboxylate ribosyl 5-

phosphate. Catalyzed by carboxylase.

 Does not require the vitamin biotin or ATP.

 Aspartate condenses with the aminoimidazole carboxylate ribosyl 5- phosphate to form

aminoimidazole 4- succinylcarboxamide ribosyl 5-phosphate. Catalyzed by synthetase.
 Adenosuccinatelyase cleaves off fumarate & only the amino group of aspartate is retained to
yield aminoimidazole 4-carboxamide ribosyl 5- phosphate.

 N10-Formyl tetrahydrofolate donates one carbon moiety to produce 5- formaminoimidazole

4- carboxamide ribosyl 5- phosphate. Catalyzed by formyltransferase.

 The final reaction catalyzed by cyclohydrolase leads to ring closure with an elimination of
water molecule.

 The product obtained is Inosine Monophosphate (IMP), the parent purine nucleotide from
which other purine nucleotides can be synthesized.
REGULATIONS OF NUCLEOTIDES METABOLISM

Purine Nucleotide Biosynthesis Is Regulated by Feedback Inhibition

Three major feedback mechanisms cooperate in regulating the overall rate of de novo purine
nucleotide synthesis and the relative rates of formation of the two end products, adenylate and
guanylate . The first mechanism is exerted on the first reaction that is unique to purine synthesis:
transfer of an amino group to PRPP to form 5-phosphoribosylamine. This reaction is catalyzed
by the allosteric enzyme glutamine-PRPP amidotransferase, which is inhibited by the end
products IMP, AMP, and GMP. AMP and GMP act synergistically in this concerted inhibition.
Thus, whenever either AMP or GMP accumulates to excess, the first step in its biosynthesis from
PRPP is partially inhibited.

In the second control mechanism, exerted at a laterstage, an excess of GMP in the cell inhibits
formation of xanthylate from inosinate by IMP dehydrogenase, without affecting the formation
of AMP. Conversely, an accumulation of adenylate inhibits formation of adenylosuccinate by
adenylosuccinate synthetase, without affecting the biosynthesis of GMP. When both products are
present in sufficient quantities, IMP builds up, and it inhibits an earlier step in the pathway; this
regulatory strategy is called sequential feedback inhibition. In the third mechanism, GTP is
required in the conversion of IMP to AMP, where as ATP is required for conversion of IMP to
GMP (Fig. 22–36), a reciprocal arrangement that tends to balance the synthesis of the two
ribonucleotides.

The final control mechanism is the inhibition of PRPP synthesis by the allosteric regulation of
ribose phosphate pyrophosphokinase. This enzyme is inhibited by ADP and GDP, in addition to
metabolites from other pathways for which PRPP is a starting point.
Figure 1 : Regulatory mechanisms in the biosynthesis of adenine and guanine nucleotides
in E. coli.

Pyrimidine Nucleotide Biosynthesis Is Regulated by Feedback Inhibition

Regulation of the rate of pyrimidine nucleotide synthesis in bacteria occurs in large part through
aspartate transcarbamoylase (AT Case), which catalyzes the first reaction in the sequence and is
inhibited by CTP, the end product of the sequence (Fig. 22–38). The bacterial ATCase molecule
consists of six catalytic subunits and six regulatory subunits (see Fig. 6–33). The catalytic
subunits bind the substrate molecules, and the allosteric subunits bind the allosteric inhibitor,
CTP. The entire ATCase molecule, as well as its subunits, exists in two conformations, active
and inactive. When CTP is not bound to the regulatory subunits, the enzyme is maximally active.
As CTP accumulates and binds to the regulatory subunits, they undergo a change in
conformation. This change is transmitted to the catalytic subunits, which then also shift to an
inactive conformation.
DISORDERS OF PURINES CATABOLISM

Various genetic defects in PRPP synthetase (reaction ➀, present clinically as gout. Each defect
—for example, an elevated Vmax, increased affinity for ribose 5-phosphate, or resistance to
feedback inhibition—results in overproduction and over excretion of purine catabolites. When
serum urate levels exceed the solubility limit, sodium urate crystalizes in soft tissues and joints
and causes an inflammatory reaction, gouty arthritis. However, most cases of gout reflect
abnormalities in renal handling of uric acid. While purine deficiency states are rare in human
subjects, there are numerous genetic disorders of purine catabolism. Hyperuricemias may be
differentiated based on whether patients excrete normal or excessive quantities of total urates.
Some hyperuricemias reflect specific enzyme defects. Others are secondary to diseases such as
cancer or psoriasis that enhance tissue turnover.

Lesch-Nyhan Syndrome

The Lesch-Nyhan syndrome, an overproduction hyperuricemia characterized by frequent

episodes of uric acid lithiasis and a bizarre syndrome of self-mutilation, reflects a defect in
hypoxanthine-guanine phosphoribosyl transferase, an enzyme of purine salvage. The
accompanying rise in intracellular PRPP results in purine over production. Mutations that
decrease or abolish hypoxanthine-guanine phosphoribosyl transferase activity include deletions,
frame-shift mutations, base substitutions, and aberrant mRNA splicing.

Von Gierke Disease

Purine overproduction and hyperuricemia in von Gierke disease (glucose- 6-phosphatase

deficiency) occurs secondary to enhanced generation of the PRPP precursor ribose 5-phosphate.
An associated lactic acidosis elevates the renal threshold for urate, elevating total body urates.

Hypouricemia
Hypouricemia and increased excretion of hypoxanthine and xanthine are associated with a
deficiency in xanthine oxidase, EC 1.17.3.2 due to a genetic defect or to severe liver damage.
Patients with a severe enzyme deficiency may exhibit xanthinuria and xanthine lithiasis.
Adenosine Deaminase & Purine Nucleoside Phosphorylase Deficiency

Adenosine deaminase deficiency is associated with an immunodeficiency disease in which both

thymus-derived lymphocytes (T cells) and bone marrow–derived lymphocytes (B cells) are
sparse and dysfunctional. Patients suffer from severe immunodeficiency. In the absence of
enzyme replacement or bone marrow transplantation, infants often succumb to fatal infections.
Defective activity of purine nucleoside phosphorylase (EC 2.4.2.1) is associated with a severe
deficiency of T cells, but apparently normal B-cell function. Immune dysfunctions appear to
result from accumulation of dGTP and dATP, which inhibit ribonucleotide reductase and thereby
deplete cells of DNA precursors.

PYRIMIDINE CATABOLITES ARE WATER SOLUBLE

Unlike the low solubility products of purine catabolism, catabolism of the pyrimidines forms
highly water-soluble products—CO2, NH3, β-alanine, and β-aminoisobutyrate. Excretion of β-
amino isobutyrate increases in leukemia and severe x-ray radiation exposure due to increased
destruction of DNA. However, many persons of Chinese or Japanese ancestry routinely excrete
β-aminoisobutyrate.

Disorders of β-alanine and β-aminoisobutryrate metabolism arise from defects in enzymes of

pyrimidine catabolism. These include β-hydroxybutyric aciduria, a disorder due to total or
partial deficiency of the enzyme dihydro pyrimidine dehydrogenase, EC 1.3.1.2. The genetic
disease reflects an absence of the enzyme. A disorder of pyrimidine catabolism, known also as
combined uraciluria-thyminuria, is also a disorder of β-amino acid metabolism, since the
formation of β- alanine and of β-aminoisobutyrate is impaired. When caused by a genetic error,
there are serious neurologic complications. A non genetic form is triggered by the administration
of the anticancer drug 5-fluorouracil (see to patients with low levels of dihydropyrimidine
dehydrogenase.

Orotic Aciduria

The orotic aciduria that accompanies the Reye syndrome probably is a consequence of the
inability of severely damaged mitochondria to utilize carbamoyl phosphate, which then becomes
available for cytosolic 824 overproduction of orotic acid. Type I orotic aciduria reflects a
deficiency of both orotate phosphoribosyltransferase (EC 2.1.3.3) and orotidylate decarboxylase,
EC 4.1.1.23 (reactions ⑤ and ➅. The rarer Type II orotic aciduria is due to a deficiency only of
orotidylate decarboxylase (reaction ➅).

Deficiency of a Urea Cycle Enzyme Results in Excretion of Pyrimidine

Precursors

Increased excretion of orotic acid, uracil, and uridine accompanies a deficiency in liver
mitochondrial ornithine transcarbamoylase (see reaction ②, Excess carbamoyl phosphate exits to
the cytosol, where it stimulates pyrimidine nucleotide biosynthesis. The resulting mild orotic
aciduria is increased by high-nitrogen foods.

Drugs May Precipitate Orotic Aciduria

Allopurinol, an alternative substrate for orotate phosphoribosyltransferase (reaction ⑤,

competes with orotic acid. The resulting nucleotide product also inhibits orotidylate
decarboxylase (reaction ➅, resulting in orotic aciduria and orotidinuria. 6-Azauridine,
following conversion to 6-azauridylate, also competitively inhibits orotidylate decarboxylase
(reaction ➅, enhancing excretion of orotic acid and orotidine. Four genes that encode urate
transporters have been identified. Two of the encoded proteins are localized to the apical
membrane of proximal tubular cells.

References
Lehninger Principles of Biochemistry (Sixth Edition). David L. Nelson Professor of
Biochemistry University of Wisconsin–Madison Michael M. Cox Professor of
Biochemistry University of Wisconsin–Madison

Harper’s Illustrated Biochemistry 31st Edition.