BIOCHEM EXAM 4 - MASTER

Where does catabolism of dietary proteins begin?

In the stomach, through the action of pepsin (enzyme that hydrolyzes proteins in a nonspecific manner).

Pepsin

Enzyme that hydrolyzes proteins in a nonspecific manner. Active enzyme but synthesized and released into the stomach as the inactive zymogen, pepsinogen.

Pepsinogen

Inactive zymogen that becomes pepsin when it is activated (cleaved) due to the low pH environment in the stomach.

Autocatalytic enzyme

An enzyme, like zymogen, that can clip residues out of itself to form the active enzyme.

Catabolism of dietary proteins after the stomach

Continues in the duodenum (the first area of the small intestine). Occurs via the action of pancreatic proteases that are produced as zymogens in the pancreas and then are released into the duodenum.

How do pancreatic proteases cleave proteins?

At specific residues as part of a proteolytic cascade that begins through the action of enteropeptidase that is found on the surface of the enterocytes (epithelial cells that line the lumen of the small intestine).

What does enteropeptidase activate?

The pancreatic protease cascade that turns zymogens into active enzymes.

What do the active enzymes activated by enteropeptidase do?

Cleave proteins to short oligopeptides, which are further cleaved via the action of amino peptidase to form dipeptides and tripeptides, which are then internalized.

Enzymes secreted in the pancreas which are activated through trypsin-initiated proteolysis

Chymotrypsinogen, Elastase, Carboxypeptidase, Lipase.

Why does bicarbonate enter the duodenum with pancreatic proteolytic zymogens?

Bicarbonate raises the pH of the stomach, inactivating pepsin and preventing enzymes from being randomly cleaved in the small intestine.

Reasons for endogenous protein catabolism

Regulation through turnover, quality control, and for a source of amino acids.

Where do cellular proteins go for degradation?

They are first tagged by the ubiquitin pathway, and then degraded by the proteasome.

How does ubiquitin bind to a protein?

Through the formation of an isopeptide bond.

What are the parts of the proteasome?

The whole proteasome is 26S. The 19S units are the caps, and 20S region is that catalytic region that degrades the protein.

How does the 20S region of the proteasome degrade proteins?

Threonines at the N-terminal ends of the subunits within the central core of the enzyme attack the protein backbone. Peptides are cleaved into short fragments that average 7-9 amino acids in length.

How are peptide fragments further broken down?

Into amino acids, which can either be left intact for biosynthesis or broken down.

What happens when amino acids are broken down?

The amino groups are given off, which can be disposed of by the urea cycle. The carbon skeletons are used to make glucose or glycogen, or to make fatty acids, or they can be oxidized completely in the TCA cycle.

Where does amino acid catabolism primarily occur?

In the liver, although the muscles break down branched-chain amino acids.

What are the branched chain amino acids?

Leucine, isoleucine, and valine.

Problems with ammonium in the blood

Ammonium is highly toxic, and high levels in the blood can cause lethargy, gait disturbances, headache, vomiting, seizures, and cognitive deficit.

What is ammonium used for?

The synthesis of nitrogen-containing compounds. Excess ammonium is converted to urea in the urea cycle.

Basic catabolic progression of amino groups

(1) Amino transfer to an alpha-keto acid.
(2) Oxidative deamination to remove the amino group as an ammonium ion.

What is left after transdeamination of amino groups?

A carbon skeleton, which can be used as an anabolic precursor or for energy production.

Transamination reaction for amino catabolism

The amino group from aspartate is transferred to the a-ketoglutarate, forming OAA and glutamate.

What prosthetic group do all aminotransferases require?

Pyridoxal phosphate, which is derived from vitamin B6.

How does pyridoxal phosphate work during the aminotransferase reaction:

Pyridoxal Phosphate binds to the amino group of the incoming amino acid to make a protonated Schiff base. Then the carbon skeleton is hydrolyzed off and leaves the aminotransferase.

Two important types of transaminases

(1) Aspartate transaminase (AST). Amino group from aspartate is transferred to alpha-ketoglutarate to yield OAA and glutamate.
(2) Alanine transaminase (ALT). Alanine transfers amino group to alpha-ketoglutarate to yield pyruvate and glutamate. Part of the alanine cycle.

Glucose-alanine cycle

An important cycle that the muscles use when they're required to exercise even when there isn't very much glucose in the blood. Muscles break down glycogen and branched-chain amino acids to yield energy.

Mechanism for the glycogen catabolism in the muscles

Glycogen is broken down to Glucose 1-Phosphate, then Glucose 6-Phosphate, then pyruvate.

Mechanism for the glucose-alanine cycle in the muscles

Muscle cells cannot safely eliminate nitrogen as urea, so amino groups are transferred to pyruvate which makes alanine, and then alanine goes through the blood to the liver.

Mechanism for glucose-alanine cycle in the liver

Once in the liver, alanine is transaminate by passing its amino group to alpha-ketoglutarate to yield glutamate. The amino group can then by hydrolyzed off, and used in the urea cycle to be eliminated safely. Once alanine loses the amino group, it becomes pyruvate, which can be used in gluconeogenesis.

Amino acids that can be directly deaminated

Serine and Threonine.

What enzymes catalyze direct deamination of amino acids?

Dehydratases

What is the last step before the urea cycle in oxidative deamination?

The removal of the amino group from glutamate to yield alpha-ketoglutarate via the action of glutamate dehydrogenase.
(1) Oxidation to form an aldimine Schiff-Base (requires NAD+ or NADP+)
(2) Hydrolysis to remove free ammonium from alpha-ketoglutarate.

Glutamate dehydrogenase

A mitochondrial enzyme that uses NAD+ or NADP+ to oxidize glutamate to form a Schiff-base intermediate as part of oxidative deamination.

Elimination of nitrogen in mammals

Amino acids give off amino group to leave an alpha-keto acid, and then amino group is transferred to alpha-ketoglutarate to make glutamate. This amino group is then released through oxidative deamination. This is packaged into urea, and is excreted.

Ureotelic

Animals, such as mammals and most other terrestrial animals, that excrete nitrogen as urea.

Uricotelic

Animals, such as birds and reptiles, that excrete nitrogen as uric acid.

Ammonioitelic

Animals, like fish, that excrete nitrogen as ammonium, as is.

Ketogenic only amino acids

Both L's: Leucine and Lysine.

Amino Acids that are both ketogenic and glucogenic

Phenylalanine, Isoleucine, Tryptophan, and Tyrosine. (Phil is tripping on tires).

Amino acids catalyzed specifically due to association with inborn errors of metabolism

Phenylalanine, Tyrosine, Valine, Leucine, and Isoleucine.

Catabolism of phenylalanine

Feeds directly into the catabolism of tyrosine.
(1) Hydroxylation to make tyrosine, catalyzed by phenylalanine hydroxyls.
(2) Deamination to form hydrophenylpyruvate.
(3) Formation of homogentisate.
(4) Homogentisate deoxygenate makes 4-Maleylacetoacetate.
(5) This is broken down to Fumarate and Acetoacetate.

Phenylketonuria

Autosomal recessive disease that results from a mutation in phenylalanine hydroxyls or insufficiency concentrations of tetrahydrobiopterin. Causes severe neurological impairments. Treated by diet low in phenylalanine and high and tyrosine.

Alkaptonuria

Absence or deficiency of the enzyme homogentisate oxygenate. Causes urine to be black. Mostly a benign disease.

Catabolism of branched-chain amino acids

Occurs in muscles in order to generate energy.
1) Transamination catalyzed by the branched-chain amino acid transaminase, where alpha-ketoglutarate is the amino group acceptor. Yields glutamate and three alpha-ketoacids.
(2) Oxidative decarboxylation of the alpha-ketoacids via action of the branched-chain alpha-ketoacid dehydrogenase complex to yield three different CoA derivatives.

Maple Syrup Urine Disease

Caused by defect in the branched-chain alpha-ketoacid dehydrogenase complex. Results in a build up of alpha-ketoacids and can leave to failure to thrive.

What is the nitrogen source for the planet?

Atmospheric nitrogen gas.

What must occur before nitrogen can be used?

Must be split and reduced to make ammonium. Most of the newly fixed nitrogen (about 60%) is produced by archaea and bacteria).

Diazotrophic Organisms

Organisms like some bacteria and archaea that can split and reduce atmospheric nitrogen, reducing it to ammonia.

Requirements for Nitrogen splitting

The triple nitrogen bond is difficult to break, so this requires special catalysts (enzymes) found in higher organisms.

Reaction for Nitrogen splitting

N2 + 10 H+ + 8 e- → 2 NH3+ + H2
Requires 16 ATP per mole of N2. Occurs using the reductase (Fe) and Nitrogenase (MoFe) portions of the protein.

Mechanism for Nitrogen fixation

(1) Reductase: transfers electrons from a donor protein (like ferredoxin) to nitrogenase.
(2) Nitrogenase: splits and reduces nitrogen.
The enzyme halves are brought together through the hydrolysis of ATP.
This reaction is energetically downhill overall, but intermediates are high in energy and unusable.

What type of bacteria fix Nitrogen?

(1) Free living bacteria, such as bacteria that live in soil.
(2) Symbiotic bacteria, such as those in root nodules and legumes. Here, leghemoglobin binds to O2, and NH3 is produced, not H2O.
Nitrogenase Complex

How does the process of incorporating Nitrogen react to oxygen?

Nitrogen is very sensitive to oxygen, so this must be kept out of the system. Leghemoglobin sequesters oxygen.

Major enzymes in the Nitrogen flow

(1) Aspartate Amino Transferase: reversible
(2) Alanine Amino Transferase: reversible
(3) Glutamine Synthetase: irreversible formation of Gln
(4): Glutaminase: Irreversible Loss of Gln
(5) Glutamate Dehydrogenase: in animals, loss of NH4+

Glutamate Dehydrogenase (in prokaryotes)

Alpha-Ketoglutarate and Ammonia combine to form Glutamate, using NAD(P)H. This reaction releases water. A Shiff base intermediate is formed.

Glutamine Synthetase

Adds another amino group to Glutamate.
(1) Glutamate is phosphorylated by ATP to make an acyl-phosphate intermediate.
(2) This intermediate is amidated, releasing Pi and forming Glutamine.

How is Glutamine Synthetase regulated?

Sensitive to regulation by negative feedback from Alanine and Glycine. Most amino acid pathways are regulated in this way.

What pathways provide metabolic intermediates to make amino acids?

Glycolysis, the Pentose Phosphate Pathway, and the TCA cycle.

What are the ten essential amino acids?

PVT TIM HALL. Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Histidine, Arginine, Leucine, Lysine.

Kwashiorkor

A disease where there are not enough amino acids in the diet to support life.

What pathways are amino acids important precursors in?

The synthesis of nucleotides, phospholipids, neurotransmitters, and hormones.

Glutathione

An antioxidant made up of glutamate, cysteine, and glycine.

How is ammonium transported from in the peripheral tissues?

Primarily as glutamine, and as alanine from the muscles.

What are the three major systems that release ammonium?

(1) Liver: NH4+ goes to the urea cycle.
(2) Kidney: NH4+ is excreted in the urine.
(3) Brain: sensitive to NH4+ and glutamate.

Enzymes used in the Liver to remove ammonium

Glutamine synthetase: present in NH4+ scavenging cells where the urea cycle is not present, which protects the body.
Glutaminase: present in cells with the urea cycle, produces urea.

Enzymes used in the Kidneys to remove ammonium

Glutaminase: glutamine is deaminated to glutamate.
Glutamate Dehydrogenase: glutamate is deaminated to alpha-ketoglutarate.
NH4+ is release in the urine.

Enzymes used to reduce ammonium in the Brain

Glutamate is used as a neurotransmitter and effects neuronal function. Glutamine is non-excitatory.
Astrocytes support neuronal fiction by using glutamine synthetase to decrease the amounts of ammonium and glutamate in the brain.

Functions of Nucleotides

(1) Precursors for nucleic acid synthesis (DNA, RNA)
(2) ATP is the prime energy source for biological processes.
(3) Nucleotides can activate biomolecules for synthetic reactions.
(4) Nucleotides and derivatives are important parts of many second messenger systems.
(5) ATP is a source of phosphate for kinase reactions.

What are nucleotides made up of?

(1) Sugar (either ribose or deoxyribose).
(2) Nitrogenous base (either a purine or a pyrimidine).

What is the difference between a nucleotide and a nucleoside?

A nucleotide has one, two, or three phosphates attached. A nucleoside does not.

What is the ribonucleoside and ribonucleotide (5' monophosphate) for the base Adenine in RNA and DNA?

RNA: Adenisone and Adenylate (AMP).
DNA: Deoxyadenosine and deoxyadenylate (dAMP).

What is the ribonucleoside and ribonucleotide (5' monophosphate) for the base Guanine in RNA and DNA?

RNA: Guanosine and Guanylate (GMP).
DNA: Deoxyguanosine and Deoxyguanylate (dGMP).

What is the ribonucleoside and ribonucleotide (5' monophosphate) for the base Cytosine in RNA and DNA?

RNA: Cytidine and Cytidylate (CMP).
DNA: Deoxycytidine and Deoxycytidylate (dCMP).

What is the ribonucleoside and ribonucleotide (5' monophosphate) for the base Uracil in RNA?

Uridine and Uridylate (UMP).

What is the ribonucleoside and ribonucleotide (5' monophosphate) for the base Thymine in DNA?

Thymidine and Thymidylate (TMP).

What are the two categories of nitrogenous bases?

Purines: Adenine and Guanine (these have two rings).
Pyrimidine: Cytosine, Uracil, Thymine (these have one ring).

What are the two ways to synthesize nucleotides?

(1) Salvage pathway: recycled bases.
(2) De Novo pathway: making bases.

Salvage pathway for nucleotide synthesis

Activated ribose (PRPP) + base → Nucleotide

De novo pathway for nucleotide synthesis

Activated ribose (PRPP) + amino acids + ATP + CO2 + ... → Nucleotide

Salvage pathway mechanism for nucleotides

5-phosphoribosyl 1-pyrophosphate + guanine → guanosine monophosphate + pyrophosphate.
Reaction driven forward by the conversion of PPI → 2Pi

De novo pathway for Pyrimidines

Pyrimidine rings are simple, formed first, and then brought to the activated ribose platform PRPP to make UTP. UTP can be used to make CTP which is used to make RNA. UTA is also used to make TTP and CTP can be used to make dCTP, both of which are used to make DNA.

De novo pathway for Purines

Purine rings are formed on the activated ribose sugar, piece-by-piece.

Steps to the pyrimidine de novo synthesis pathway

(1) Orate synthesis (carbamoyl phosphate and aspartate)
(2) Oritdylate formation (attachment of orate to PRPP)
(3) UMP formation (decarboxylation)
(4) UTP (phosphorylation)
(5) CTP (amination)
(6) TPP and dCTP (reduction)

Synthesis of carbamoyl phosphate in the pyrimidine de novo pathway

Bicarbonate → carbamoyl phosphate.
This uses 2 ATP and one NH3 to release 2 ADP and one Pi. The enzyme used is Carbamoyl Phosphate Synthetase II. This is the rate-limiting step in pyrimidine de novo synthesis.

Where does the amino group come from in the formation of carbamoyl phosphate?

Ammonium or glutamine. The reaction is as follows:
Gln + CO2 + 2ATP + H2O → carbamoyl phosphate + Glu + 2ADP + Pi.
Reaction catalyzed by carbamoyl phosphate synthetase II.

How does the carbamoyl phosphate synthetase II enzyme work?

Works by channeling, which diminishes the loss of substrate by diffusion. This helps to prevent nonproductive reactions such as the hydrolysis of ATP.

Where does carbamoyl phosphate go after formation?

Combines with Aspartate to form Orotate using the enzyme Aspartate Transcarbamoylase. This reaction releases water and requires an NAD+.

Where does orotate go after formation?

Orotate is added to PRPP using the enzyme pyrimidine Phosphoribosyltransfers, forming Orotidylate (OMP). This reaction is driven forward by the release of PPi.

Where does Orotidylate go after synthesis?

Decarboxylation of Orotidylate yields the uracil triphosphate precursor, Uridylate (UMP).

How is CTP made from UMP?

(1) Two kinase reactions go from UMP to UDP to UTP.
(2) An amino group from glutamine is added, which is transferred from UTP to make CTP. This process is thus an amination.

How is the pyrimidine de novo pathway regulated?

The enzyme carbamoyl phosphate synthetase catalyzes the rate-limiting step, and is inhibited by UTP and activated by PRPP.
The enzyme aspartate transcarbamolyse is negatively regulated by CTP and positively regulated by ATP..

What is the first committed step in purine synthesis?

PRPP + NH3 → 5-Phosphoribosyl-1-amine + PPi.
Addition of ammonia from glutamine to PRPPR to form 5-Phosphoribosyl-1-amine. Catalyzed by the enzyme glutamine phosporibosyl amidotransferase.

Regulation of glutamine phosphoribosyl amidotransferase

Allosteric regulation by downstream products. AMP/ADP, GMP/GDP.

Where does phosphoribosylamine go after synthesis in the purine de novo pathway?

This becomes Inosinate, which is a common intermediate in synthesis of AMP and GMP.
Inosinate is also called IMP, or inosine monophosphate.

What can IMP form in the de novo synthesis of purines?

Can become Adenylate (AMP), or Guanylate (GMP).

How is AMP formed from IMP?

IMP gains an amino group from Aspartate and a phosphate group from GTP. Fumarate is lost.

How is GMP formed from IMP?

IMP gains an amino group from ammonia (from a Glutamine source) and a phosphate group from ATP.

How is purine synthesis regulated?

(1) Formation of PRPP inhibited by IMP, AMP, and GMP.
(2) Formation of Phosphoribosylamine inhibited by IMP, AMP, and GMP.
(3) Formation of Adenylosuccinate inhibited by AMP.
(4) Formation of Xanthylate inhibited by GMP.