BCH4024 Exam 2 Nitrogen Metabolism

primary metabolites

necessity for normal operation of metabolic pathways and main cellular functions

ie Amino acids, nucleotides, RNA, DNA, B vitamins

secondary metabolites

not needed for cell growth, development, or reproductions

ie alkaloids in plants

amino acid sources

dietary proteins, endogenous protein turnover, and de novo synthesis

dietary proteins

supply essential and non essential amino acids, humans can only make 10

intracellular proteolysis

removes misfolded, old and damaged proteins, regulates cell metabolism and important in cell cycle transitions, also known as endogenous protein turnover

de novo synthesis

needed to adjust AA levels within tissues and for protein synthesis; able to synthesize specialized AAs, controls leval of central pathway metabolites

Provides nutritionally nonessential amino acids that are needed for protein synthesis Adjusts amino acid pools in different tissues Adjusts energy metabolism by controlling concentrations of central pathway metabolitesAllows cells to adapt to metabolic stress Needed to make nucleotides, heme, hormones, as well as neurotransmitters.

essential AAs

histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine

PVTTIMHLL; variety needed in diet!

conditionally essential AAs

arginine in pregnant women/children

tyrosine when Phe is low

Cystine when Met is low

intracellular proteolysis

breaking down and building new proteins in cells; not at a constant rate, Low Nitrogen intake leads to increased of this; 3 pathways are lysosomal, proteasome, and autophagic

lysosome pathway

proteins protonated in lysosome with low acidic pH and undergo partial unfolding making them susceptible to proteolysis

proteasome pathway

protein ubiquitin marks unfolded proteins for breakdown, only ubiquinated proteins can enter proteasomes, barrel like macromolecule strcutruers that use proteases to form small peptides and AAs

autophagic pathway

removes old organelles ie mitochondria/ribosomes via engulfing in vacuole using ubiquitin+ lysosomes

ubiquitination

post translational modification that marks proteins using 3 enzymes, E1/E2/E3, initiating conjugating and ligase,

mono and poly; mono adds to linked molecules and poly adds to N terminus of ubiquitin

digestion

mechanical and enzymatic within GI; HYDROLYTIC process as water cleaves functional groups; biopolymers myst be broken down into monomers so done so via this route;

enzymes of this are produced/stored in pancreas and stomach and neurohormonally controlled

proteolysis

enzymatic cleaves of proteins; 2 steps by breaking protein into fragments and then into AAs and di/tri peptides

protein fragment proteolysis

occurs in small intestine at neutral pH where protein fragments can not refold; done by chymotrypsin, trypsin, carboxypeptidase, and elastase- cleaves in CT

protein proteolysis

saliva- proteases from bacteria/WBCs

stomach- acidic pepsin

zymogen

protease enzymes as inactive precursors made/stored in stomach/pancreas which secretes into small intestine; safer to store and prevents autophagy and apoptosis

ie pepsinogen, chymotrypinogen, trypsinogen, pro-carboxypeptidase

pepsinogen

pepsin, stomach (gastric chief cells), ph 1-3; autocatalytics, 2 active site aspartyl residues use to mediate low pH catalyssis

gastric chief cells also excrete chymosin, an acid protease that speeds up pepsin cleavage by curdling milk

chymotrypsinogen

active trypsin cleaves this then also pi chymotrypsin+ remaining trypsinogen to activate sequential proteolysis

trypsinogen

made in pancreas and stores in vesicles, TI keeps in inactive form, in small intestine TI is diluted so enterokinase activates this to turn into trypsin

trypsin is used for activation of chymotrypsionogen

enterokinase

ectoprotease on intestinal mucosal wall

pro-carboxypeptidase

cleaved by trypsin

AA transporters

symporter imports AAs/ sodium into intestinal brush cells against AA gradient; driven by transmembrane ion gradient because of high sodium content in intestine lumen and low sodiium on brush border cells

sodium gradient maintained by sodium potassium ATPase; maintains chemiosmotic concentration gradient

chemi= using energy of ATP hydrolysis

osmotic= imbalance of ions on different sides

true nitrogen balance

intake=excretion; de novo synthesis important in maintaining N balance, different across orgnas

positive nitrogen balance

intake exceeds excretions; required for growth in children/pregnancy, wound healing and convalescence

negative nitrogen balance

intake less than excretion; starvation, malnutrition, disease ie burns, trauma surgery

marasmus

Protein energy malfunction ie PEM from inadequate protein and calorie intake, deficency of all nutrients; dry skin and loose folds of skin hanging over butt,

SUPER SKINNY

kwashiorkor

protein malnutrition but getting carbs so fat; irritability, enlarged liver, edema, osmotic imbalance

big stomach, washing of african fruit to lose nutrients

transamination

conversion of keto-acids and AAs through loss of alpha amino group via double displacement by transaminases; alpha keto acids essential for metabolism in right quantiy ie not too much or too little

transaminases

requires cofactors PLP derived from vitamin B6 and bind extremely tight; PLP converted to PMP

Pyridoxal phosphate/PLP

pyridoxamine phosphate/pmp

pyruvate

alpha keto acid of alanine

oxaloacetate

alpha keto acid of ASP

alpha ketoglutarate

alpha keto acid of glutamate

transamination mechanism

2 half reactions, half 1: formation of aldimine, then conversion to ketimine which is hydrolyzed to ketoacid ;half 2: occurs in reverse with R2

fullreversible

AAs that do not undergo transamination

proline/hydroxyproline: secondary amines no transamination

lysine: would cyclize into toxic non metabolite

threonine: dimerize into toxic non metabolite

transaminases cannot bind lysine or threonine

oxidative deamination

done by glutamate dehydrogenase GDH to regenerate alpha-ketoglutarate, the amino acceptor and prodduces ammonia for reutilization or disposal; located in mitochondrial matrix

GDH mechanism

amine oxidized to imine with NAD+ reduction, imine then hydrolyzed to keto acid; NADH formed as well

transaminase and deamination can be coupled to oxidative degrade 14 AAs

NAD in GDH

converted to NADH; creates large energy storage release if C-N bond broke, enough energy to make 3 ATP; nature’s batteries; eaction runs on dependence of availability of NAD/NADPH, NO NADH OR NADP+

when high levels of NAD+ mitochondria favor oxidative deamination and GDH catalyzes oxidation; makes atp

when high levels of NADPH, favor reductive amination, GDH catalyzes reduction; used to make fatty acids and sterols

GDH products

a-KA enters citric acid cycle, NH4+ to urea cycle, NADH reoxidized to NAD by oxidative phosphorylation ie making ATP;

GDH activity

dependent on Energy state of cell and available AAs ie allosteric control; when

HIGH ATP/GTP/NADH; GDH IS INHBITED, so low rate of AA degradation and high protein synthesis, deamination favored at lower pH

HIGH ADP/ GDP/ FREE AAs, GDH is highly active, a-KA stimulates TCA to make ATP, fuels ETS and oxidative phosphorylation restoring energy and amination favored at HIGH pH

routes of Deamination

GDH main system for removing ammonia from AAs

can also be hydrolytic or eliminative

L/D AA oxidases

similar to GDH ie converts AA to a-KA; H2O2 produced and flavin mononucleotides used as cofactors; humans make both L and D

D AAs found in damaged protein and old, dry food

D- AA oxidase has antibacterial effect by preventing accumulation of D AAs

hydrolytic deamination

glutaminase- widely dsitributed in body in mitochondria; ammonia enters urea cycle, also done by

asparaginase

eliminative deamination

histidinase or histidine ammonia lysae

ammonia assimilation

incorporating of ammonia into metabolic form, toxic at high concentration so must be kept at 5-10 micromolar; free ammonia can cross membranes and dissipate proton gradient

free ammonia depletes a-Kglutatarate inhibiting TCA activity

high ammonia in mitochondria of liver because urea is produced here

ammonia assimilation pathways

GDH, Glutamine synthetase, carbamoyl synthetase, glycine synthetase

GDH in ammonia

reductive amination, reverse of oxidative amination reaction, no effect on NAD/NADH

NADPH + NH3 + α-KG → Glutamate + H2 O + NADP+

glutamine synthetase

main way of trapping ammonia, GLN serves as major N donor in biosynthetic reactions and is major inter-organ N shuttle; doesnt alter blood pH; allows for production of ammonia in situ which is then used as Nuc

Glutamine synthetase mechanism

double displacement via 2 SN2 rxns;

GLE phosphorylated by ATP to give gamma glutamyl P intermediate which reacts with NH3 to produce GLN

pH less than pka so mostly NH4+; deprotonation reaction driven by free energy of ATP ie Delta G = +10

Futile cycle prevention

keeping enzymes in separate cell compartments ie GLN synthetase and glutaminase; synthetase only in cytoplasm where glutaminase is in mitochondria

Carbamoyl Phosphatase Synthetase-I/CPS-1

main reaction for ammonia assimilation in mitochondria; bicarbonate phosphorylated by ATP to give C-Pi intermediate which reacts with NH3 to produce to Carbamate which is then phosphorylated

requires 2 ATP; associated with urea cycle

Carbamoyl Phosphatase Synthetase-II/CPS-2

GLN dependent enzymel GLN used as N source; occurs in cytosol using GLN and aprotic transfer tunnel allows for free NH3 with no energetic cost to deprotonate

CPS-2 mechanism

GLN- hydrolyzing subunit initially filled with water which is displaced by GLN, water is w5 so too big; hydrolyzes GLN to release NH3 into tunnel while a gamma thioester is formed and acts as lid to block proton entry

ammonia moved unprotonated through tunnel between active sites where it goes to react with intermediate

GLN dependent enzyme reactions have low michaelins constant for GLN, high affinity

liver cells

show division of labor ie carry out different metabolic tasks in different organelles or subcellular locations

metabolite pool- cells make and store metabolites where needed, pool is reservoir of metabolite; carbamoyl P has 2 destinies in liver

urea cycle basics

form in which N is excreted, varies between species ie

fish excrete NH3- ammonotelic

Birds excrete uric acid- uricotelic

mammals excrete urea- ureotelic

90-95% of N excreted in urea form; excretion depends on daily N intake in dietl some excretion via creatinine and uric acid; gout= excess uric acid

urea cycle

occurs almmost exclusively in liver ie principal NH3 detoxification site, N transported between organs as ALN, GLE, GLN, urea; enzymes in cytosol and mitochondria; this cycle liberates considerable acid helping control body pH

urea cycle mechanism

ornithine+CP1→ citruline

citruline+ aspartate→ arginosuccinate

arginosuccinate→ arginine+ fumarate

arginine→ ornithine +urea

step 1 urea cycle

ornithine nuc amine group attacks carbamoyl- P to make citruline; requires ortnithine transcarbamoylase OTC, ornithine comes from GLN semi aldehyde

step 2 urea cycle

citruline + asp make arginosuccinate;

REQUIRES ATP AND ARGINOSUCCINATE SYNTHASE

step 3 urea cycle

breakdown of argininosuccinate into arginine and fumarate using argininosuccinate lyase

fumarate can be converted to oxaloacetate or ASP and enter TCA

step 4 urea cycle

argninine breakdown into ornithine and urea via ARGINASE and a hydrolysis recation; ornithine returns to mitochondria to restart urea cycle; urea excreted

overall reaction

(HCO3-)+ (NH4+)+(3ATP)+(ASP)+(H2O)→ Urea +(2ADP)+(2Pi)+ AMP+ PPi+ Fumarate+ (5H+)

the big picture

lots of protons are released helping to control body pH, carbonate comes from TCA, NH3 from GDH, ATP from oxidative phosphorylation

Urea cycle control

high protein diet stimulates urea cycle enzymes, AAs caused glucagon release inducing enzyme biosynthesis, low proein diet not a lot of urea, certain AAsa re effective in stimulating glucagon release ie casein, decreases NH3 thus increasing OAA and a-KG for gluconeogenesis

glucagon

pancreatic alpha cell hormone that stimulates glucose synthesis

indirect activation of urea cycle

ARG + GLE important regulators due to role in NAG production (N-acetylglutamate); R allosterically stimulates conversion and NAG synthase, E is substrate for synthesis

NAG

allosertic activator of CPS1, the first step of the urea cycle; deficient leads to hyperammonemia, primary deficiency is mutation in NAG gene,

secondary deficiency= mitochondrial changes affecting NAG function

NAG anolgue N-carbamoylglutmate (carbaglu) is used when deficiencies exist AND activates CPS1

OTC deficiency

ornithine transcarbamoylsae, most common inherited urea cycle disorder, X-linked in adult of any age or sex, rapid NH3 increase leading to lethargy and potential death

arginase deficiency

episodic hyperammonia if untreated, not life threatening, plasma R levels 3-4x normal, birth and early childhood levels are normal because R required for growth

liver acinus

averting ammonia toxcity by prevents NH3 re-entry into blood through differential use of NH3 , hepatocytes in direct contact with blood to permit rapid metabolite exchange, early cells in first segment of acinar space are rich in glutaminase and urea cycle enzymes, high km=low affinity for NH3, removes majority of NH3, perivenous scavenger cells are rich in GLN synthetase, low km, high affinity nH3, mops up leftover NH3 and supplies GLN to many organs

endothelial lining of liver is fenestrated exposing to hepatocytes permitting rapid metabolite exchange between liver and blood

biosynthesis of arginine

key urea cycle intermediate, diet, cell protein turnover, and ornithine; can be converted to nitric oxide and citrulline; can be used to synthesize creatine; nitric oxide helps protect tissues from damage due to low blood supply

creatinine synthesis

creatine is converted to a phosphorylated form in muscle using creatine kinase and then creatinine without enzyme; produced at fairly constant rate bc no enzyme; production amount dependent on muscle amount so higher in men; clearance rate is indication of how well kidney functions

biosynthesis of glutatamate

transamination; reductive amination ie GDH, reduction amination GLN synthetase, GLN hydrolysis with glutaminase

biosynthesis of Glutamine

only GNL synthetase, cytosolic enzyme

biosynthesis of Aspartate

transamination, hydrolysis ie asparaginase

biosynthesis of asparagine

asp hydrolysis, uses 2 active sites and transfer tunnel

biosynthesis of alanine

transamination only; used for inter-organ transport btwn liver and organs of ammonia nitrogen to prevent toxicity, “alanine shuttle”, alanine→circulation→ alanine

biosynthesis of proline

reduction of E to E semi-aldehyde, cyclase, then reduced again, note that this semi aldehyde can undergo transamination to make ornithine

biosynthesis of serine

2 pathways beginning with 3-Pi-glycerate; made from glycolytic intermediate

biosynthesis of glycine

4 pathways, simplest structure yet hardest to make

biosynthesis of tyrosine

conditionally essential, requires Phe; 2 step catalytic proc

biosynthesis of hydroxyproline

collagen fibril maturation- conversion within collagen, stabilizes collagen by forming key hydrogen bonds within tropocollagen triple helix, Prolyl hydroxylase is easily deactivated, forming bound Fe(III)–O – ,which must be reduced by Vitamin C to restore enzyme:; iron dependents and associated with scurvy

phenylketonuria (PKU)

deficiency in PHE hydroxylase; high serum PHE (20x normal) + high urinary phenylpyruvate upon transamination; very rare, damages nerve function but is treatable with low PHE diet that is maintained into adulthood

phenyl-pyruvate

autosomal recessive

two copies of abnormal gene must be present for the disease or trait to develop; PKU is an example

biosynthesis of cysteine

conditionally essential, requires MET, SAM is involved, a methylating agent for 300 metabolites

s-adenosylmethionine

thyroxin biosynthesis

formed on thyroglobulin; protein synthesized within thyroid cells but stores outside, iodide is actively transported into thyrocytes and oxidized to reactive iodine in ERl occurs thru EAS, forming thyronine ring, reabsorbed after CNS stimulationl protelyzed to form T4 endocrine

iodination of free tyrosine does not occur

thyronine

specialized fused ring AA, T3 is active form (10x over T4 (thyroxin))

metabolic actions of T3

t3 formed by t4 peripheral deiodinase; enters cells through active transporter binding to mobile receptors/transcription factors to stimulate or inhibit gene expression, increase basal metabolic rate

thyroid hormones improve development, growth, and metabolism but not necessity for life

triiodothyronine (t3)

AA neurotransmitters

E and N are excitatory, D-ser, gly; some are converted to GABA

GABA

gamma- aminobutyric acid; formed by decarboxylation of L- E

important neurotransmitters

D-Ser from racemization of L-Ser

serotonin from tryptophan

dihydroxyphenilalanine from PHE

pyrimidine biosynthesis

fewer AAs/steps than purine with simpler structures, ring formed first then added to PRPP, UMP initially formed which is phosphorylated to UTP

cytosine

thymine