Lecture 12: Ribonucleotide Reductase & deoxy-Nucleotide metabolism

What are the three primary cellular roles of nucleotides mentioned in the material?

Building blocks for DNA/RNA, energy transfer, and cell signaling.

The universal precursor used for the synthesis of both purines and pyrimidines is _____.

5-phospho-D-ribosyl-1(alpha-pyrophosphate (PRPP)

Which enzyme catalyzes the conversion of Ribose-5-phosphate to PRPP?

PRPP synthetase (Prs)

In de novo purine synthesis, the purine ring is constructed step-by-step on a _____ platform.

Ribose

Which amino acids provide all the nitrogen atoms for the purine ring system?

Glutamine, Glycine, and Aspartate.

What is the committed step in de novo purine biosynthesis?

The conversion of PRPP to 5-phosphoribosylamine (PRA).

Which enzyme catalyzes the committed step of purine synthesis?

Glutamine-PRPP amidotransferase.

What provides the driving force for the synthesis of 5-phosphoribosylamine (PRA)?

The hydrolysis of pyrophosphate (PP_i).

Name the common intermediate from which both AMP and GMP are derived.

Inosine Monophosphate (IMP).

Which energy-rich molecule drives the synthesis of AMP from IMP?

GTP

Which energy-rich molecule drives the synthesis of GMP from IMP?

ATP

In purine regulation, what is the effect of high GTP levels on AMP synthesis?

It promotes AMP synthesis.

How do AMP and GMP regulate their own synthesis from the branch point intermediate IMP?

Through feedback inhibition of their respective synthesis pathways.

Purinosome

A multi-enzyme complex that carries out the de novo synthesis of purine nucleotides.

Which molecules act as feedback inhibitors for PRPP synthetase?

ADP and GDP.

How does de novo pyrimidine synthesis differ from purine synthesis regarding the ribose attachment?

The pyrimidine ring is built first and then attached to the ribose platform.

Where is the enzyme Carbamoyl Phosphate Synthetase II (CPS-II) located in the cell?

Cytosol

Which three enzyme activities are contained within the single polypeptide chain known as CAD?

CPS-II, Aspartate Transcarbamylase (ATCase), and Dihydroorotase.

The bifunctional enzyme 'UMP Synthase' consists of which two activities?

Orotate phosphoribosyltransferase and Orotate decarboxylase.

In pyrimidine synthesis, which molecules provide 'substrate activation' for the CAD complex?

ATP and PRPP.

Which pyrimidine end-products inhibit the activity of the CAD complex?

UDP, UTP, and CTP.

How does GTP influence the synthesis of CTP?

GTP activates CTP Synthase to balance the CTP and GTP pools.

Which enzyme is responsible for the general reaction XDP + ATP to XTP + ADP?

Nucleoside diphosphate kinase (NDK).

The reaction AMP + ATP to 2 ADP is catalyzed by which specific kinase?

Adenylate kinase

Why does the cell typically maintain RNA building block levels 5 to 10 times higher than DNA building blocks?

Because most carbon flux in nucleotide metabolism generates ribonucleotides for short-lived RNA and energy transfer.

Deoxyribonucleotides (dNTPs) are synthesized directly from which precursors?

Ribonucleoside diphosphates (rNDPs).

What is the primary enzyme responsible for reducing ribonucleotides to deoxyribonucleotides?

Ribonucleotide Reductase (RNR).

On which carbon of the ribose sugar does RNR remove the -OH group?

The 2'carbon.

What is the ultimate reducing agent for the reduction of ribonucleotides by RNR?

NADPH

Which protein acts as the intermediate electron carrier between NADPH and RNR?

Thioredoxin

What byproduct is released during the reduction reaction catalyzed by RNR?

A water molecule (H₂O).

Why is it critical for the cell to maintain a proper balance of the four dNTPs?

Deficiency is lethal and excess can be mutagenic.

The RNR enzyme complex is composed of which two types of subunits?

The catalytic alpha2 subunits and the free radical-generating beta2 subunits.

Where are the active sites located in the Ribonucleotide Reductase tetramer?

At the interface between the alpha2 and beta2 subunits.

Which functional group in the RNR alpha subunit is essential for its catalytic activity?

Sulfhydryl groups (thiols).

What specific type of free radical is generated within the RNR beta subunit?

A tyrosyl radical.

Which metal cofactor is found within the beta2 subunits of RNR?

Binuclear iron (Fe^{3+}).

Which kinase converts dUDP, dGDP, dADP, and dCDP into their respective triphosphates?

Nucleoside diphosphate kinase (NDK).

In the CAD complex, which enzyme catalyzes the formation of carbamoyl aspartate from carbamoyl phosphate and aspartate?

Aspartate Transcarbamylase (ATCase).

What intermediate is produced by IMP Dehydrogenase during GMP synthesis?

Xanthylate (XMP).

What intermediate is formed by Adenyl-Succinate Synthase during AMP synthesis?

Adenyl-succinate

In de novo purine synthesis, which molecule provides the C2 and C8 atoms of the purine ring?

N^{10}-Formyl THF

Which molecule provides the N1 atom of the purine ring?

Aspartate

Which molecule provides the N3 and N9 atoms of the purine ring?

The amide nitrogen of Glutamine.

How many ATP molecules are consumed to synthesize one IMP molecule de novo?

4 ATPs

Which enzyme in pyrimidine synthesis is a QH2-dependent flavoprotein (FMN)?

Dihydroorotate dehydrogenase

What is the end product of the reaction catalyzed by Orotate Decarboxylase?

Uridine Monophosphate (UMP).

In pyrimidine synthesis, which molecule provides the nitrogen atom at position 1?

Aspartate

In pyrimidine synthesis, which molecule provides the nitrogen atom at position 3?

The amide nitrogen of Glutamine.

What is the approximate molecular weight of the active RNR tetramer?

approx 250 kDa

Which broad specificity kinase generates nucleoside triphosphates from diphosphates using ATP as a donor?

Nucleoside diphosphate kinase (NDK).

The synthesis of dUDP from UDP is catalyzed by _____.

Ribonucleotide Reductase (RNR)

Which molecule is the precursor of dTMP in the thymidylate synthesis pathway?

dUMP

What is the role of NADH in the synthesis of GMP?

It is a byproduct formed when IMP is oxidized to XMP by IMP Dehydrogenase.

Which amino acid is released as a byproduct when XMP is converted to GMP?

Glutamate (Glu).

In AMP synthesis, which molecule is released after the cleavage of adenyl-succinate by adenyl-succinate lyase?

Fumarate

What molecule is the phosphate donor for the conversion of GDP to GTP?

ATP

The pathway of de novo purine synthesis is described as _____ while pyrimidine synthesis is more _____.

branched; linear

What is the effect of GTP on the activity of CTP synthase?

GTP activates CTP synthase.

How many sulfur atoms are involved in the redox cycle of each alpha subunit of RNR?

Two (from two sulfhydryl groups).

In the active structure of RNR, what is the approximate distance between the Fe center and the catalytic site?

approx 35AA

Which nucleotide specifically inhibits CTP Synthase?

CTP