Final Exam Mon 8 am

Reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase.

FIGURE 22-40a Ribonucleotide reductase. (a) Subunit structure. Each active site contains two thiols and a group (—XH) that can be converted to an active-site radical; this group is probably the —SH of Cys439, which functions as a thiyl radical.

Ribonucleotide reductase. The R2 subunits of E. coli ribonucleotide reductase. The Tyr residue acts as the tyrosyl radical.

binuclear iron center

The tyrosyl radical functions to generate the active-site radical (—X ･ )

Proposed mechanism for ribonucleotide reductase. In the enzyme of E. coli and most eukaryotes, the active thiol groups are on the R1 subunit; the active-site radical (—X ･ ) is on the R2 subunit and in E. coli is probably a thiyl radical of Cys439

Regulation of ribonucleotide reductase by deoxynucleoside triphosphates. The overall activity of the enzyme is affected by binding at the primary regulatory site (left). The substrate specificity of the enzyme is affected by the nature of the effector molecule bound at the second type of regulatory site, the substrate-specificity site (right). The diagram indicates inhibition or stimulation of enzyme activity with the four different substrates.

Biosynthesis of thymidylate (dTMP). The pathways are shown beginning with the reaction catalyzed by ribonucleotide reductase.

Conversion of dUMP to dTMP by thymidylate synthase and dihydrofolate reductase.

Serine hydroxymethyltransferase is required for regeneration of the N5,N10-methylene form of tetrahydrofolate.

In the synthesis of dTMP, all three hydrogens of the added methyl group are derived from N5,N10-methylenetetrahydrofolate (pink and gray).

Catabolism of purine nucleotides.

Catabolism of a pyrimidine.

Shown here is the pathway for thymine.

The methylmalonylsemialdehyde is further degraded to succinyl-CoA.

Allopurinol, an inhibitor of xanthine oxidase. Hypoxanthine is the normal substrate of xanthine oxidase. Only a slight alteration in the structure of hypoxanthine yields the medically effective enzyme inhibitor allopurinol.

At the active site, allopurinol is converted to oxypurinol, a strong competitive inhibitor that remains tightly bound to the reduced form of the enzyme.In addition to blocking uric acid production,

inhibition of xanthine oxidase causes an increase in hypoxanthine and xanthine, which are converted to the purines adenosine and guanosine monophosphates. Increased levels of these ribotides causes feedback inhibition of amidophosphoribosyl transferase, the first and rate-limiting enzyme of purine biosynthesis. Allopurinol therefore decreases both uric acid formation and purine synthesis.

Azaserine and acivicin, inhibitors of glutamine amidotransferases. These analogs of glutamine interfere in several amino acid and nucleotide biosynthetic pathways.

Thymidylate synthesis and folate metabolism as targets of chemotherapy. During thymidylate synthesis, N5,N10-methylenetetrahydrofolate is converted to 7,8-dihydrofolate; the N5,N10-methylenetetrahydrofolate is regenerated in two steps

Thymidylate synthesis and folate metabolism as targets of chemotherapy

inhibits thymidylate synthase

inhibits dihydrofolate reductase

aminopterin, is identical to methotrexate except that it lacks the shaded methyl group

Regulatory mechanisms in the biosynthesis of adenine and guanine nucleotides in E. coli. Regulation of these pathways differs in other organisms.

Methylation of dUMP into dTMP

• Thymidylate synthase is a target for some anticancer drugs

Conversion of dUMP to dTMP by thymidylate synthase and dihydrofolate reductase.

Catabolism of Purines: Formation of Uric Acid

• Excess purine nucleotides are dephosphorylated into nucleosides and phosphate

• Adenosine yields hypoxanthine via deamination and hydrolysis

• Guanosine yields xanthine via hydrolysis and deamination

• Hypoxanthine and xanthine are oxidized into urate, the anion of uric acid

• Spiders and other arachnids lack xanthine oxidase

Catabolism of Purines: Degradation of Urate to Allantoin

• Urate is oxidized into a 5-hydroxy-isourate by urate oxidase

• Hydrolysis and the subsequent decarboxylation of 5-hydroxy-isourate yields allantoin

• Most mammals excrete nitrogen from purines as allantoin

• Urate oxidase is inactive in humans and other great apes;

we excrete urate• Birds, most reptiles, some

amphibians, and most insects also excrete urate

NH

N NH

NH

O

O

O

NH

N N

NH

O

O

O OH

NH2

NH

NH

NH

O

O

O

H

H+

-

-

O2 + H2O

H2O2

CO2

H2O

urate oxidase

spontaneous or catalyzed

urate

5-hydroxyisourate

allantoin

Catabolism of Purines: Degradation of Allantoin

• Most mammals do not degrade allantoin

• Amphibians and fishes hydrolyze allantoin into allantoate; bony fishes excrete allantoate

• Amphibians and cartilaginous fishes hydrolyze allantoate into glyoxylate and urea; many excrete urea

• Some marine invertebrates break urea down into ammonia

NH2

NH

NH

NH

O

O

O

H

NH2

NH

NH

NH2

O O

O

H

O

H+

OH

OO

NH2

NH2

ONH2

NH2

O

NH4+

H2O

H2O

2 H2O + 4 H+

2 CO2

4

allantoinase

allantoicase

urease

allantoin

allantoate

urea

ammonium cation

Chapter 22: Summary

• Some prokaryotes are able to reduce molecular nitrogen into

ammonia; understanding details of the nitrogen fixation is one of the

holy grails in biochemistry

• The twenty common amino acids are synthesized via difficult-to-

remember pathways from -ketoglutarate, 3-phospho-glycerate,

oxaloacetate, pyruvate, phosphoenolpyruvate, erythrose 4-

phosphate, and ribose-5-phosphate

• Nucleotides can be synthesized either de novo from simple

precursors, or reassembled from scavenged nucleobases

• Purine degradation pathway in most organisms leads to uric acid but

the fate of uric acid is species-specific

In this chapter, we learned that:

Nucleotide Metabolism Overview

HAT medium: Hypoxanthine, aminopterin, thymidine

Making monoclonal antibodies

HAT Medium

“Alfonse, Biochemistry makes my head hurt!!”\