biochem exam 2

What are primary metabolites?

they are needed for normal operation of metabolic pathways and main cellular functions

What are secondary metabolites?

- organic compounds NOT needed for cell growth, development, or reproduction

What are the functions of secondary metabolites?

* ward off pathogens and predators
* protect against osmotic damage
* treat various illnesses

What are some examples of keto-acids?

pyruvate, oxaloacetate, a-ketoglutarate 

Where is pyruvate found?

cytosol

Where is Oxaloacetate found?

* cytosol and mitochondria
* used in gluconeogenesis and citric acid cycle

Where is a-ketoglutarate found?

* mitochondria
* used in citric acid cycle

What is the structure of pyruvate, oxaloacetate, and a-ketoglutarate?

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What are the sources of amino acids?

* digestion of protein in food
* synthesis of amino acids
* prides aa's needed for protein synthesis
* adjust aa pools in different tissues
* adjusts energy metabolism by controlling levels of central pathway metabolites
* allow cells to adapt to metabolic stress
* needed to synthesize neurotransmitters and nucleotides

What is the use of Amino Acids?

intracellular proteolysis:

* removes misfolded proteins
* removes old and damaged proteins
* regulates metabolism
* controls cell-cycle transitions

"What are the 9 ""nutritionally essential (not made in body) amino acids?"

Phe, Val, Trp, Thr, Ile, Met, His, Leu, Lys

PVT TIM HiLL

What are the conditionally essential amino acids?

* Arg: needed for growth childhood and pregancy
* Tyr: becomes essential when Phe is inadequate
* Cys: becomes essential when Met is inadequate

What is most Met presented as?

S-Adenosyl-Met

T or F: Omnivores are disadvantaged when is comes to getting a varied balance of amino acids in their diet.

F: they are advanataged

What is found in the 3 areas of proteolysis (enzymatic cleavage of proteins)?

* Saliva: proteases principally from bacteria and white blood cells
* stomach: pepsin
* intestine: neutral proteases like chymotrypsin (cleaves on the carboxyl end of aromatic residue), trypsin (cleaves on carboxyl end of lysine and arginine residues), carboxypeptidases (cleaves C-terminal AA), Elastase (cleaves elastin)

What is the path for proteolysis?

proteins --> stomach --> proteins that can't refold --> intestine -->  amino acids + Di- and Tri-peptides 

T or F: dietary proteins are absorbed by healthy intestines

F: must be proteolyzed to amino acids, di and tri- peptides 

What begins enzymatic proteolysis?

making inactive proteases (zymogen)

\- synthesized and stored in pancreas

\- secreted into small intestine

\- converted to active catalysts

What happens in trypsinogen activation?

* Pancreas makes and stores trypsinogen in vesicles
* secretory vesicles contain trypsin inhibitor (revents unwanted proteolysis of host cells)
* enterokinase, an ectoprotease on intestinal mucosal wall, converts trypsinogen into trypsin
* trypsin activates chymotrypsinogen 

What is Pro-carboxypeptidase?

* activated to carboxypeptidase
* removes amino acids one-by-one from C-termini of food proteins

Where does zymogen synthesis, processing, and transport occur?

ribosomes attached to rough ER -> golgi complex 

Where does zymgen vesicale targeting and release occur?

zymogen granule

T or F: Zymogen formation prevents autophage and apoptosis 

T

Where is pepsinogen formed in and released by?

gastric chief cells within the stomach

What does it mean by pepsinogen activation is autocatalytic?

once a little active pepsin forms, the latter catalyzes cleavage of many pepsinogen molecules to form more catalytically active enzyme molecules

How does pepsin operate at low pH?

by using its aspartic acid carboxyl groups for catalysis (aspartic proteinase)

T or F: turnover rate depends on metabolic state.

T: greater protein degradation, when nitrogen intake is low, because cells need amino acids to make vital proteins (ex: antibodies and hormones like insulin)

What is the lysosomal/phagolysosomal pathway?

* lysosome is an acidic compartment, where proteins undergo isoelectric expansion (partial unfolding
* low pH makes them more susceptible to proteolysis 

What is true nitrogen balance? positive? negative?

* True: intake = excretion
* Positive: intake > excretion
* Negative: excretion > intake

What is positive nitrogen balance required for?

* growth in childhood
* growth in pregnancy
* healing of wounds
* convalescence 

When does negative nitrogen balance occur?

* starvation
* malnutrition
* disease (burns, trauma, surgery)

What is marasmus?

* malnutrition associated with extensive tissue and muscle wasting, with little/no edema
* protein-energy malfunction resulting from inadequate intake of proteins and calories
* severe deficiency of nearly all nutrients, especially protein, carbohydrates, and lipids"

What is Kwashiorkor?

* sickness last baby gets when the new baby arrives
* acute childhood protein malnutrition
* unlike marasmus, kwashiorker has inadequate protein intake, BUT otherwise adequate caloric intake
* charactertistics: irritability (neurotransmitter deficit), enlarged liver (fatty infiltrates), edema (caused by hypoalbuminemia)"

Transaminases use \________ coenzymes

Vitamin B6

transaminases catalyze 2 half reactions

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What is the mechanism of enzyme-bound reactions?

* 1st half-reaction: aldimine forms; converts to ketimine; hydrolyzes to ketoacid
* 2nd half-reaction: reverse of 1st half reaction, using R2-KA to make R2-AA

aldimine vs ketamine

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What is the Keq for transamination?

Keq= 1 (same bonding in substrates and products)

\-fully reversible

alanine: alpha- ketogluterate transaminase

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glutamate: oxaloacetate transaminase

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Which 4 amino acids CANNOT undergo transamination?

* proline and hydroxyproline: don't have the needed primary amine for transamination
* lysine: if transaminated, the keto acid would cyclize to form a toxic nonmetabolite
* threonine: if transaminized, the keto acid would dimerize into a toxic nonmetabolite

T or F: transaminases cannot bind prolines and hydroxyprolines.

F: cannot bind lysine or threonine

What is the reaction equation foer glutamate dehydrogenase?

* major route for oxidative deamination
* glutamate + NAD^+  + H2O

What is the structure for a-ketoglutarate?

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Where is glutamate dehydrogenase (GDH) located?

mitochondrial matrix

What are nature's batteries?

* NAD+ and NADH
* NADH oxidation yields enerfy needed to make 3 mol ATP from ADP and Pi through oxidative phosphorylation

What are the exceptions to the coupling of GDH with transaminases allowing for oxidative degradation of 14 other amino acids?

Pro, Hyp, Thr, Lys, His

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memorize

How does GDH use NAD+ in oxidative deamination reaction?

* NADH is re-oxidized to NAD+ in oxidative phosphorylation
* a-KA1 enters citric acid cycle
* excess NH4+ enters urea cycle.
* operating in the **opposite** direction, 
* NADPH drives reductive amination to form glutamate.

GDH uses what?

NAD+and NADPH (NOT NADH and NADP+)

What happens when the mitochondria rapidly re-oxidizes NADH to NAD+?

* excess NAD+
* NAD+>>NADH
* NADH oxidation makes ATP in Mitochondria

What happens when there is excess NADPH?

* NADPH>>NADP+
* NADPH is used to make fatty acids, sterols, etc.

What inhibits GDH?

* high ATP, GTP, and NADH
* reduces AA degradation and favors protein synthesis

What activates GDH?

* high ADP, GDP, and free amino acids
* a-KG stimulates citric acid cycle, fueling ATP synthesis

Glutaminase catalyzes \__________?

hydrolysis of glutamine 

What is the glutaminase reaction equation?

glutamine + H2O ----> glutamate + NH3

Histidinase (or Histadine ammonia lyase) catalyzes \_________? 

deamination of Histidine 

histidine

Why do we need both L- and D- Amino acid oxidases?

* there are D- amino acids in damaged proteins
* D- amino acids are also abundant in old and dried food
* by forming ketoacids from D-AA, D- amino acid oxidase allows cells to harvest carbon skeletons as an energy source 

Asparaginase catalyzes \_____________?

hydrolysis of asparagine

asparagine -----> Aspartate + NH3

What are the 4 enzymes that catalyze NH3 assmilation?

* Glutamate Dehydrogenase:
* NADPH + NH3 + a-KG ---> Glutamate + H2O + NADP+


* Glutamine Synthetase:
* ATP + NH3 + Glutamate ----> Glutamine + Pi + ADP + H+


* Carbamoyl-Phosphate Synthetase I:
* 2 ATP + NH3+ CO2 ----> H2N(C=O)-O-P+ Pi + 2ADP + H+

What is Glutamine Synthetase?

* Main way to trap NH3
* Gln is a major nitrogen source
* Gln is a major inter-organ nitrogen shuttle, avoiding direct transfer of NH3 from distant organs to liver

What are the 2 SN2 reactions that occur in sequence for glutamine synthetase?

Glutamate ----ATP ADP---> gamma-Glutamyl-P (essential intermediate) ----> Glutamine

What drives ammonium ion deprotonation?

* ∆GATP-hydrolysis
* pK of ammonium ion deprotonation= 9.2
* ∆Gdeprotonation= 10 kJ/mol
* driven by ATP-dependent conformation change that moves guanidium group of a nearby arginine residue near enzyme-bound NH4+ to allow electrostatic repulsion to form NH3

What would happen if opposing enzymes were kept in the same compartment?

* futile cycles would pointlessly hydrolyze ATP
* Glutamine synthetase would convert glutamate to glutamine and glutaminase would convert glutamine to glutamate in a continuous cycle

What is Carbamoyl-Phosphate Synthetase- I?

* main ammonia-assimilating reaction in mitochondria


* highly energy-dependent reaction


* 1st reaction resembles glutamine synthetase reaction and second reaction is a phosphorylation reaction

Why is glutamine vital for the efficient biosynthesis of nitrogen-containing metabolites?

* allows glutamine-dependent biosynthetic enzymes to generate ammonia *in situ*
* that ammonia is then used as a nucleophile without ever touching water

What is CPS II? 

* glutamine dependent


* uses a transfer tunnel to move unprotonated NH3 from glutamine-hydrolysis site to biosynthetic site


* covalently bound glutamate acts as a ""lid"" covering the glutamine site, preventing entry of H2O or H3O+


* used throughout metabolism

Which subunit in CPS-II hydrolyzes Gln to Glu + NH3?

glutamine-hydrolyzing subunit

T or F: The use of CPS-II allows are body to hydrolyze glutamine to deprotonate NH3 exactly where and when it is needed to avoid toxicity of ammonia elsewhere in out cells.

T

What are the characteristics of glutamine-dependent biosynthetic enzymes?

* exhibits a low Km (high affinity) for glutamine
* essential -SH group generates a gamma-glutamyl thioester that accupies active site, permitting NH3 transfer
* two active sites connected by ammonia transfer tunnel that prevents protonation of NH3 even at neutral pH

What are the series of reactions that represent glutamine-dependent biosynthetic enzymes?

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What is the differences between CPS I and CPS II?

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What is the structure of Uric Acid? What is the structure of ammonia?

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What do fish excrete?

NH3 - Ammonotelic

What do birds excrete?

Uric Acid - Uricotelic

What do mammals excrete?

Urea - Ureotelic

What is gout?

Diseased caused by creating too much uric acid

What is the principal site of NH3 detoxification?

* liver


* high levels of ammonia in the blood is toxic so it is transported in organic forms like alanine, glutamate, glutamine, and urea

What is the overall stoichiometry for urea synthesis?

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T or F: Urea cycle is an energy-independent pathway which liberates considerable acid to help control body pH.

F: Urea cycle is energy-dependent 

What is the reaction equation for ornithine transcarbamoylase? Reaction mechanism for argininosuccinate synthase? 

* Reaction: Aspartate + Gln + ATP

What is the structure for ornithine, citrulline, and arginino-succinate?

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What is the reaction equation for argininosuccinate lyase?

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Reaction equation for arginase? 

What is the Urea Cycle?

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How does a high-protein diet induce urea cycle enzymes?

* certain amino acids stimulate glucagon release (casein or milk protein is rich in certain AAs)
* glucagon stimulates biosynthesis of urea cycle enzymes
* reduces ammonia load and increases pyruvate, OAA, and aKG for glucogenesis.

How do arginine and glutamate regulate the urea cycle?

* CPS-I is the 1st commited step of the Urea Cycle
* CPS-I is allosterically activated by N-acetylglutamate.
* When amino acid catabolism increases, glutamate levels rise and so does N-acetyl-glutamate.

What is the reaction equation for NAGS (N-Acetyl-Glutamate Synthase)?

Glutamate + Acetyl CoA <\---> N-Acetyl-Glutamate + CoA

How are glutamine and arginine related to amino acid content and NAG?

* Glu and Arg are indicators of high amino acid content
* Glu is substrate for NAG synthase
* Arg activates NAGS allosterically 
* NAG synthase deficiency results in hyperammonemia 

How does the liver acinus avery ammonia toxicity?

* liver acinus is an ammonia trap
* endothelial lining within the liver is fenestrated (full of openings, exposing hepatocytes to blood)
* hepatocytes are in direct contact with blood components which permits rapid metabolite exchange between liver and blood

What does the liver acinus do?

- prevents ammonia re-entry into circulation through differential utilization of ammonia 

Cells in the first segment of the acinar space contain \____________?

* glutaminase and urea cycle enzymes
* CPS-I has low affinity for NH3
* Urea cycle removes bulk of NH3
* Low affinity, High Capacity

After the blood goes through the first segment of the acinar space, blood passes through \____________, which are rich in \______________.

* perivenous scavenger cells; glutamine synthetase


* GS gas 40x higher affinity for NH3


* GS mops up NH3 that gets past the first segment


* Supplies glutamine to many organs


* High affinity, Low capacity

How is Arginine synthesized?

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What is creatinine?

* break-down product of creatine-P in our muscles
* produced at a fairly constant uncatalyzed rate
* yield depends on muscle mass; higher in men
* clearance rate tells us how well kidney is working

glutamate synthesis

transamination → reductive amination →hydrolysis

how is glutamine made?

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What enzyme exclusively makes glutamine?

Glutamine synthetase (located in cytosol)

aspartate synthesis

transamintion → hydrolysis

What is the reaction for Asparagine synthesis? Explain the reaction.

* Reaction: Aspartate + Gln + ATP