Synthesis and Degradation of Nucleotides

Part 1: September 1st, 2009

Champion CS DeivanayagamCenter for Biophysical Sciences and EngineeringUniversity of Alabama at BirminghamBirmingham, AL 35294-4400

Information Transfer in Cells

The fundamental process of information transfer in cells.

Purines and Pyrimidines

Note that the numbering are slightly different particularly where the glycosidic bonds are attached

Gylocosidic bondGylocosidic bond

Nucleotide: purines and pyrimides linked to a ribose/de-oxy ribose sugar moiety

Two types of pathways leads to nucleotides:

1. De novo pathway – Begins with their metabolic precursors: amino acids, ribose-5-phosphate, and CO2 – The free bases A, T, G, C, and U are not intermediates – Appears to be present in identical form in nearly all living organisms

2. Salvage pathway – Recycle the free bases and nucleosides released from nucleic acid breakdown – The free bases are intermediates

I. De Novo Purine Nucleotide Synthesis

The initially synthesized purine derivative is inosine.

Purines are initially formed as ribonucleotides rather than as free bases

AMP (Adenosine monophosphate)

GMP (Guanosine monophosphate)

John Buchanan (1948) "traced" the sources of all nine atoms of the purine ring

Origin of the nine atoms:

Bird feed containing selectively labeled atoms

Examination of the isotope distribution in excreted uric acid (dove poop research!!!)

• N-1 from aspartic acid • N-3, N-9 from glutamine • C-4, C-5, N-7 from glycine • C-6 from CO2 • C-2, C-8 from THF - one carbon units

11 steps lead to the formationOf IMP (Ionosine mono-phospate)

The names of the enzymes can be a mouthful !!! In addition each of these enzymes have AKA’s (common name and EC names)

It would be great if you could memorize them, however it is not necessary to memorize all the steps in this reaction

What you need to learn from this lecture ?

1. What are the Committed steps that are unique in this synthesis cycle2. What are the different feed back inhibition steps in this synthesis cycle3. What steps can be utilized to develop inhibitors in this synthesis cycle4. What are some of the diseases that are related to this synthesis cycle

A committed step is an effectively irreversible reaction in the biosynthesis pathway

Glutamine PRPP amidotransferase is subject to feedback inhibition

GMP, GDP, GTP as well as AMP, ADP and ATP carry out this action.

How? Through an allosteric site present on the enzyme.The G series of nucleotides at a Guanine-specific allosteric site on the enzyme

andThe A series of nucleotides at an Adenine-specific allosteric site on the enzyme

Glutamine PRPP amidotransferase is also inhibited by ‘azaserine’

Azaserine acts as an irreversible inhibitor of glutamine-dependent enzymes by covalently attaching to nucleophilic groups in the glutamine-binding site. It is used as an anti-tumor agent.

Allosteric enzyme cartoon representation

(aka FGAM synthetase)

(aka Adenylosuccinate lyase)

(aka IMP cyclohydrolase)

In vertebrates these reactions are coupled together

One multifunctional polypeptide chain (110 kda) encodes for:GAR synthetase (step 3)GAR tranformylase (step 4)AIR synthetase (step 6)

Another encodes forAIR carboxyalse (step 7)SACAIR synthetase (step 8)

Another polypeptide chain 67 kD (organized as 135 kDa dimers)AICAR tranformylase (step 10)IMP synthase (step 11)

Folate Analogs as Antimicrobial and Anticancer Agents

• De novo purine biosynthesis depends on folic acid compounds at steps 4 and 10

• For this reason, antagonists of folic acid metabolism indirectly inhibit purine formation and, in turn, nucleic acid synthesis, cell growth, and cell development

• Rapidly growing cells, such as infective bacteria and fast-growing tumors, are more susceptible to such agents

• Sulfonamides are effective anti-bacterial agents

• Methotrexate and aminopterin are folic acid analogs that have been used in cancer chemotherapy

Step 12: Synthesis of Adenine and Guanine Ribonucleotides:

a). AMP is made from IMP in two steps.The first step converts IMP to adenylosuccinate. The second step is catalyzed by adenylosuccinate lyase that produces AMP.

b). The formation of GMP from IMP requires oxidation at C-2 of the purine ring, followed by a glutamine-dependent amidotransferase reaction that replaces the oxygen on C-2 with an amino group to yield 2-amino, 6-oxy purine nucleoside monophosphate – i.e., GMP. The second reaction is catalyzed by GMP synthetase, shown here.

The Purine Biosynthetic Pathway is Regulated at Several Steps

Allosteric regulation occurs in the first two steps, and AMP and GMP are competitive inhibitors in the two branches at right.

Can Cells Salvage Purines?

• Nucleic acid turnover (synthesis and degradation) is an ongoing process in most cells

• Purines that are obtained from diet and turn over and not degraded can be reconverted to nucleosided tri-phosphates and be reused.

• Nucleotides are then converted to nucleosides by specific nucleotidases and non-specific phosphotases

NMP + H2O nucleoside + Pi

• Nucleosides are hydrolyzed by nucleosidases or nucleoside phophorylases to release the purine base

Nucleoside + H2O Nucleosideases base + ribose Nucleoside + Pi Nucleoside phosphorylase base + ribose-1-P

• Salvage pathways collect hypoxanthine and guanine and recombine them with PRPP to form nucleotides in the HGPRT reaction

Major pathways of purine catabolism in animals.

Major pathways of purine catabolism in animals.

Animals Oxidize Uric Acid to Different Excretory Products

HGPRT Converts Bases Back to Nucleotides

Notice: PRPP is also involved in the salvage pathway.

HGPRT - Hypoxanthine-guanine phosphoribosyltransferase

Lesch-Nyhan Syndrome – HGPRT Deficiency Leads to a Severe Disorder

Victims of Lesch-Nyhan syndrome experience severe arthritis due to accumulation of uric acid, as well as retardation, and other neurological symptoms.

Lesch-Nyhan syndrome results from a complete deficiency in HGPRT.

Absence of HGPRT is the cause of Lesch-Nyhan syndrome

In L-N, purine synthesis is increased 200-fold and uric acid is elevated in blood

Major pathways of purine catabolism in animals.

Lack of Adenosine Deaminase is One Cause of Severe Combined Immunodeficiency Syndrome

SCID is a group of related disorders involving diminished immune responses. 30% of SCID patients lack the enzyme adenosine deaminase. In the absence of ADA, deoxyadenosine is not deaminated to deoxyinosine as normal (above).

Gout is a Disease Caused by an Excess of Uric Acid

• Xanthine oxidase (XO) in liver, intestines (and milk) can oxidize hypoxanthine (twice) to uric acid

• Humans and other primates excrete uric acid in the urine, but most N goes out as urea

• Birds, reptiles and insects excrete uric acid and for them it is the major nitrogen excretory compound

• Gout occurs from accumulation of uric acid crystals in the extremities • Precipitation and deposition of uric acid causes arthritic pain and kidney stones• Causes: impaired excretion of uric acid and deficiencies in HGPRT

Allopurinol, an analog of hypoxanthine, is a potent inhibitor of xanthine oxidase.

Allopurinol binds tightly to xanthine oxidase, preventing uric acid formation. Hypoxanthine and xanthine do not accumulate to harmful concentrations because they are more soluble and thus more easily excreted.

Summary of disorders of Purine Metabolism:

Disorder Defect Comments

Gout PRPP synthase/ Hyperuricemia HGPRT

Lesch Nyhan lack of HGPRT Hyperuricemia syndrome

SCID ADA High levels of dAMP

von Gierke’s disease glucose 6-phosphatase HyperuricemiaReading assignment:

Tomorrow:

1. Pyrimindine synthesis/degradation2. Deoxyribonucleotide synthesis