Lecture 37: Porphyrins and Nucleotides

What are the molecules derived from amino acids?

• biological amines

  • neurotransmitters like dopamine and epinephrine from Tyr

  • histamine from His

  • serotonin from Trp

• other hormones and signaling molecules

  • thyroid hormone from Tyr

  • NO from Arg

• polymers

  • melanin pigment from Tyr

  • plant lignin from Phe

• porphyrins (heme)

• nucleotides

What are porphyrins?

planar, cyclic compounds with four pyrrole rings joined through methylene bridges

What does porphyrin readily bind?

metal ions such as Fe²⁺ and Fe³⁺

Where are porphyrins found?

• hemoglobin

• myoglobin

• cytochromes

• catalase

What is the building block for synthesizing porphyrins?

gamma-aminolevulinate

Describe how gamma-aminolevulinate is formed.

reaction 1:

• substrate(s): succinyl-CoA and glycine

• cofactor(s): PLP

• enzyme: gamma-aminolevulinate synthase

• product(s): a-amino-B-ketoadipate and CoA-SH

reaction 2:

• substrate(s): a-amino-B-ketoadipate

• cofactor(s): PLP

• enzyme: gamma-aminolevulinate synthase

• product(s): gamma-aminolevulinate and CO₂

In mammals, what is gamma-aminolevulinate synthesized from?

glycine and succinyl-CoA

In bacteria and plants, what is gamma-aminolevulinate synthesized from?

glutamate

Describe the synthesis of porphyrin.

General:

1. gamma-amino levulinate (GAL)

2. porphobilinogen

3. preuroporphyrinogen

4. uroporphyrinogen III

5. coproporphyrinogen

6. protoporphyrinogen

7. protophoyrin

8. heme

reaction 1:

• substrate(s): gamma-amino levulinate and 8 H₂O

• cofactor(s): none

• enzyme: porphobilinogen synthase

• product(s): porphobilinogen

reaction 2:

• substrate(s): porphobilinogen

• cofactor(s): none

• enzyme: uroporphyrinogen synthase

• product(s): preuroporphyrinogen and 4 NH₄⁺

reaction 3:

• substrate(s): preuroporphyrinogen

• cofactor(s): none

• enzyme: uroporphyrinogen III cosynthase

• product(s): uroporphyrinogen III and H₂O

reaction 4:

• substrate(s): uroporphyrinogen III

• cofactor(s): none

• enzyme: uroporphyrinogen decarboxylase

• product(s): coproporphyrinogen III and 4 CO₂

reaction 5:

• substrate(s): coproporphyrinogen III

• cofactor(s): none

• enzyme: coproporphyrinogen oxidase

• product(s): protoporphyrinogen and 2 CO₂

reaction 6:

• substrate(s): protoporphyrinogen

• cofactor(s): none

• enzyme: protoporphyrinogen oxidase

• product(s): protoporphyrin

reaction 7:

• substrate(s): protoporphyrin

• cofactor(s); none

• enzyme: ferrochelatase

• product(s): heme and Fe²⁺

True or False: The steps in porphyrin biosynthesis are common to all organisms.

true

What is the last step of porphyrin biosynthesis?

insertion of Fe²⁺ by ferrochelatase

What are prophyrias?

hereditary deficiencies in heme biosynthesis

What are the symptoms of porphyrias?

• discolored urine

• skin lesions (photosensitivity)

• anemia

• organ damage

What do macrophages convert heme to?

bilirubin

True or False: Bilirubin is carried by serum albumin in blood to the liver.

true

What color is biliverdin?

green

What happens to bilirubin in the liver?

it is conjugated to glucuronate to become more water-soluble

Where is bilirubin excreted?

in the bile (feces and urine)

What color is urobilin?

yellow

What color is stercobilin?

red-brown

What is the heme degradation process?

General:

1. heme is broken down into bilirubin in macrophages

2. unconjugated bilirubin is transported to the liver

3. bilirubin is conjugated with glucuronic acid

4. conjugated bilirubin is secreted into bile and sent to the intestine

5. glucuronic acid is removed by bacteria

6. resulting bilirubin is converted to urobilinogen

7. some urobilinogen is reabsorbed from the gut and enters the blood

8. a portion of urobilinogen enters the enterohepatic urobilinogen cycle

9. some urobilinogen is transported to the kidney, converted to urobilin, and excreted in the urine

10. the rest of the urobilinogen is oxidized by intestinal bacteria to stercobilin, which is excreted in the feces

1. senescent red cells are a major source of hemeproteins

2. breakdown of heme to bilirubin occurs in macrophages of the reticuloendothelial system (tissue macrophages, spleen, and liver)

3. unconjugated bilirubin is transported through the blood (complexed to albumin) to the liver

4. bilirubin is taken up via facilitated diffusion by the liver and conjugated with glucuronic acid

5. conjugated bilirubin is actively secreted into bile and then the intestine

6. in the intestine, glucuronic acid is removed by bacteria; the resulting bilirubin is converted to urobilinogen

7. some of the urobilinogen is reabsorbed from the gut and enters the portal blood

8. a portion of this urobilinogen participates in the enterohepatic urobilinogen cycle

9. the reminder of the urobilinogen is transported by the blood to the kidney, where it is converted to yellow urobilin and excreted, giving urine its characteristic color

10. urobilinogen is oxidized by intestinal bacteria tot he brown stercobilin

What is jaundice?

a characteristic yellow color to skin, nail beds, and sclerae (whites of the eyes)

What are the 3 types of jaundice?

• hemolytic jaundice

• hepatocellular jaundice

• neonatal jaundice

What does jaundice result from?

an accumulation of excess bilirubin in the blood, owing either to increased bilirubin production or to decreased ability of the liver to process it

True or False: Excess bilirubin accumulates in (stains) connective tissues. Jaundice is a sign of an underlying disorder (often, liver failure).

true

Neonatal jaundice is most pronounced in which individuals?

premature infants

Describe neonatal jaundice.

causes:

• hepatic bilirubin glucuronyl-transferase levels are low at birth

• enzymes may not be able to keep up with bilirubin production

• excessively high levels of bilirubin can escape from alubimin

can cause:

• neurologic damage

treatment:

• exposure to blue light converts bilirubin into more water-soluble derivatives which are excreted into bile without conjugation to glucuronate

True or False: Serum levels of bilirubin rise after birth in full-term infants, although usually not to dangerous concentrations.

true

What are the various roles for nucleotides?

• DNA and RNA synthesis

  • dNTPs and NTPs

• energy currency

  • ATP and GTP

• adenosine nucleotides in coenzymes

  • NAD, FAD, CoA

• signaling molecules

  • cAMP and cGMP

• intermediates in certain biosynthetic reactions

What are the two pathways needed for nucleotide biosynthesis?

de novo synthesis and salvage pathways

Describe de novo synthesis.

• reactions are nearly identical in all species

• different pathways for purines (A & G) and pyrimidines (C & T)

What are purine bases built up from?

from precursors on a ribose phosphate “scaffold”

How are pyrimidine bases synthesized?

pyrimidine base/orotic acid is synthesized first and attached to ribose phosphate

What are purine rings assembled on?

5-phosphoribosyl-1 pyrophosphate (PRPP)

Which precursors is PRPP assembled from? (i.e. where do the various atoms come from?)

• aspartate gives a N atom

• formate gives 2 C atoms

• CO₂ gives a C atom

• glycine gives 2 C atoms and a N atom

• the amide of glutamine gives 2 N atoms

Which pathway does PRPP come from?

the pentose phosphate pathway (PPP)

What is the first committed step of purine biosynthesis?

glutamine-PRPP amidotransferase

Describe how AMP is produced.

General:

1. PRPP

2. 5-phosphoribosylamine

3. ……

4. IMP, GTP, and aspartate

5. adenyl succinate, GDP, and P_i

6. AMP and fumarate

reaction 1:

• substrate(s): PRPP

• enzyme: glutamine-PRPP amidotransferase

• product(s): 5-phosphoribosylamine

reaction 10:

• substrate(s): IMP, GTP, and aspartate

• enzyme: adenyl succinate synthetase

• product(s): adenyl succinate, GDP, and P_i

reaction 11:

• substrate(s): adenyl succinate

• enzyme: adenylosuccinate lyase

• product(s): AMP and fumarate

Describe how GMP is produced.

General:

1. PRPP

2. 5-phosphoribosylamine

3. IMP and NAD⁺

4. XMP, Gln, and ATP

5. GMP, Glu, AMP, and PP_i

reaction 1:

• substrate(s): PRPP

• enzyme: glutamine-PRPP amidotransferase

• product(s): 5-phosphoribosylamine

reaction 10:

• substrate(s): IMP and NAD⁺

• enzyme: IMP dehydrogenase

• product(s): XMP, H₂O, NADH, and H⁺

reaction 11:

• substrate(s): XMP, Gln, and ATP

• enzyme: XMP-glutamine amidotransferase

• product(s): GMP, Glu, AMP, and PP_i

In inosinate (IMP) synthesis, which step(s) need formyl H₄ folate as a 1-carbon donor?

• GAR transformylase

• AICAR transformylase

What’s different about AIR carboxylase in IMP synthesis?

this carboxylase isn’t biotin-dependent

How does IMP synthesis differ in bacteria and mammals?

AIR can be converted to CAIR by AIR carboxylase in mammals, but this process occurs in two steps in bacteria

In IMP synthesis, which two enzymes do bacteria use in place of the AIR carboxylase that is used in mammals?

• N⁵-CAIR synthetase

• N⁵-CAIR mutase

What is the nitrogen donor in AMP and GMP synthesis?

aspartate and glutamine, respectively

Where does pyrimidine synthesis occur?

in the cytoplasm

Describe how CTP is made.

General:

1. aspartate and carbamoyl phosphate

2. N-carbamoylaspartate

3. L-dihydroorotate

4. orotate and PRPP

5. oridylate (OMP)

6. uridylate (UMP)

7. UTP, Gln, and ATP

8. CTP, Glu, ADP, and P_i

reaction 1:

• substrate(s): aspartate and carbamoyl phosphate

• enzyme: aspartate trans-carbamoylase

• product(s): N-carbamoylaspartate and P_i

reaction 2:

• substrate(s): N-carbamoylaspartate

• enzyme: dihydroorotase

• product(s): L-dihydroorotate and H₂O

reaction 3:

• substrate(s): L-dihydroorotate and NAD⁺

• enzyme: dihydroorotate dehydrogenase

• product(s): orotate, NADH, and H⁺

reaction 4:

• substrate(s): orotate and PRPP

• enzyme: orotate phosphoriboysl-transferase

• product(s): orotidylate (OMP) and PP_i

reaction 5:

• substrate(s): orotidylate

• enzyme: orotidylate decarboxylase

• product(s): uridylate (UMP) and CO₂

reaction 6:

• substrate(s): UMP and 2 ATP

• enzyme: kinases

• product(s): UTP and 2 ADP

reaction 7:

• substrate(s): UTP, Gln, and ATP

• enzyme: cytidylate synthetase

• product(s): CTP, Glu, ADP, and P_i

Where does carbamoyl phosphate come from?

the urea cycle

True or False: UMP cannot be directly converted to CMP. Instead, UMP is converted to UTP, and then into CTP.

true

Describe how nucleotides are converted into deoxynucleotides.

• substrate(s): ribonucleotide diphosphate

• cofactor(s): NADPH and H⁺

• enzyme: ribonucleotide reductase

• product(s): deoxyribonucleotide diphosphate, NADP⁺, and H₂O

Why is the conversion of ribonucleoside diphosphate to deoxyribonucleoside diphosphate considered an unusual reaction?

• reaction occurs at a non-activated carbon atom

• active-site free radical X abstracts H atom from C-3

• C radical at C-3 promotes ejection of -OH from C-2

True or False: Oxidized ribonucleotide reductase enzyme is regenerated by reduced glutaredoxin or thioredoxin (small cytoplasmic proteins).

true

True or False: Ribonucleotide reductase utilizes free radicals.

true

Describe how dTMP is produced.

• dUMP is methylated by thymidylate synthase to generate dTMP

• role of methylene tetrahydrofolate

Tetrahydrofolate is a cofactor in the synthesis of what?

• amino acids

• purines

• TMP

What do sulfonamides (sulfa antibiotics) do?

• competitively inhibits folic acid synthesis

• decreases nucleotide synthesis

Which enzyme does sulfonamides inhibit?

dihydropteroate synthetase

Sulfa antibiotics block folate synthesis in what?

in prokaryotes

What does methotrexate do?

competitively inhibits dihydrofolate reductase

What is methotrexate used to treat?

• psoriasis

• rheumatoid arthritis

• neoplastic diseases

In primates, what is purine degraded to and where is it excreted?

degraded into uric acid and excreted in the urine

True or False: Uric acid has limited water solubility and can precipitate/crystallize in joints and connective tissue—leading to local inflammation (gout).

true

What is used to treat gout?

allopurinol

What is allopurinol?

• is like hypoxanthine

• inhibits xanthine oxidase

• inhibits uric acid production

Mammals other than primates metabolize purine into what?

uric acid and then into allantoin for excretion into the urine

What does a deficiency in adenosine deaminase lead to what? What does this cause?

• leads to a loss of B and T lymphocytes

• causes severe combined immunodeficiency (SCID)

What does deoxyadenosine accumulation lead to?

build of dATP—inhibiting ribonucleotide reductase

What happens to the N atoms in pyrimidines?

atoms are either transferred to glutamate or released as ammonia

What are N atoms excreted as?

urea

What happens to the carbon skeletons of pyrmidines?

metabolized into succinyl-CoA

How are free purine bases salvaged?

bases are reattached to PRPP to generate nucleotide monophosphates

What are two examples of salvage pathways?

adenine phosphoriboxyltransferase

• converts adenine and PRPP into AMP and PP_i

hypoxanthine-guanine phosphoribosyltransferase

• converts hypoxanthine and PRPP into IMP and PP_i

• converts guanine and PRPP into GMP and PP_i

Which gene is the hypoxanthine-guanine phosphoribosyl transferase enzyme on?

the X-chromosome

What is Lesch-Nyhan syndrome due to?

due to a lack of the hypoxanthine-guanine phosphoribosyltransferase enzyme

What does Lesch-Nyhan syndrome lead to?

• nervous system damage

• self-mutilation

• gout