Biochemistry Exam 2 University of Florida BCH4024 Prof Dan Purich, Biochemistry Exam 2 Dr. Zeile

needed for normal operation of metabolic pathways & main cellular functions

primary metabolites

functions of secondary metabolites?

warding off pathogens/predators; protecting against osmotic damage; treating various illnesses.

_____ are essential for cells to operate efficiently; pivotal role in metabolism; however, too little - not good, & too much - also not good.

Keto acids

what are secondary metabolites?

those organic compounds not needed for cell growth, development, or reproduction.

a-ketoacid of Alanine

Pyruvate

a-ketoacid of Aspartate, draw structure

Oxaloacetate

a-ketoacid of Glutamate, draw structure

a-Ketoglutarate

3 sources of amino acids

1) Digestion of proteins in foodstuffs
2) Intracellular proteolysis
2) de novoSynthesis of amino acids

Digestion of proteins in foodstuffs

Supply both essential and non-essential AAs

Intracellular proteolysis (4 points)

-Removes misfolded proteins
-Removes old and damaged proteins
-Regulates metabolism
-Controls cell-cycle transitions

de novo Synthesis of amino acids (5 points), and what does de novo mean?

-Provides amino acids needed for protein synthesis
-Adjusts amino acid pools in different tissues
-Adjusts energy metabolism by controlling levels of central pathway metabolites
-Allow cells to adapt to metabolic stress
-Needed to synthesizeneurotransmitters&nucleotides

synthesis of complex molecules from simple molecules

Nutritionally Essential Amino Acids:

Phe Val Trp Thr Ile Met His Leu Lys

PVT TIM HiLL

Conditionally Essential Amino Acids

Arg, Tyr, Cys

Arginine

essential for growth (childhood &pregnancy)

Tyrosine

essential, when Phe is inadequate

Cysteine

essential, when Metis inadequate

Diet must be varied toget adequate balance of ___________

amino acids

___________ have advantage by eating both plant & animal protein

Omnivores

Saliva

Mainly contains proteases from bacteria & white blood cells

Proteolysis

enzymatic cleavage of proteins, protein fragements, and amino acids + Di and Tri peptides

Intestines

-Contains neutral proteases-Chymotrypsin(cleaves on Carboxy side of aromatic residues) -Trypsin(cleaves on Carboxy side of lysine and arginine residues) -Carboxypeptidase(cleaves C-terminal AA) -Elastase(cleaves elastin, a highly elastic protein found in connective tissue)

At what point in the GI tract are proteins broken into fragments?

the intestines

Chymotrypsin

cleaves on Carboxy side of aromatic residues

Trypsin

cleaves on Carboxy side of lysine and arginine residues

Carboxypeptidase

cleaves C-terminal AA

Elastase

cleaves elastin, a highly elastic protein found in connective tissue

Digestion: Enzymatic Proteolysis (3 points)

1) Process begins with inactive proteases (zymogens)
2) Trypsinogen Activation
3) Pro-carboxypeptidase

Dietary proteins are _______ absorbed by healthy intestine

NOT- must be proteolyzed to amino acids, di-& tri-peptides

Process begins with inactive proteases (3 steps)

zymogens (name for inactive protease)

1) Zymogens are synthesized & stored in pancreas
2) Zymogens are secreted into small intestine
3) Only then are they converted to active catalysts

Trypsinogen Activation (4 steps)

1) Pancreas makes & stores trypsinogen in vesicles.
2) Secretory vesicles contain trypsin inhibitor (Prevents unwanted proteolysis of host cells)
3) Enterokinase, anectoprotease on intestinal mucosal wall, converts trypsinogen into trypsin
4) Trypsin activates chymotrypsinogen

Pro-carboxypeptidase

activated to carboxypeptidase

Removes AAs one-by-one from C-termini of food proteins

Zymogen Activity

1. Zymogen Synthesis, Processing & Transport (golgi complex releases for transport)
2. Vesicle Targeting & Release (zymogen granule enters the lumen)

How are zymogens activated?

by proteolytic cleavage

_________ enzymes are safer to store

inactive

Zymogen formation prevents _________ & __________

autophagy and apoptosis

Four major digestive proteases:
Name the zymogen, enzyme, and ph

Pepsinogen (S), Pepsin, Optimally active at pH 1-3

Chymotrypsinogen (PI), Chymotrypsin, Optimally activeat pH 7

Trypsinogen (PI), Trypsin, Optimally activeat pH 7

Procarboxypeptidase (PI), Carboxypeptidase, Optimally active at pH 7

S = Stomach PI = Pancreatic secretion into Intestine

Trypsin: 1st cleavage forms _________________

imperfect activation site

Chymotrypsin (cleaves out two dipeptides(14-15 & 147-148), 2nd Cleavage creates and "_____________" that stabilizes transition-state

oxyanion hole

active α-Chymotrypsin ( _______ covalently attached chains)

three

Pepsinogen activation is _________________

autocatalytic

Pepsinogen to pepsin

Slow Acid-Catalyzed activation by stomach[H+]

Once a little active pepsin forms, the latter catalyzes cleavage of many pepsinogen molecules to formmore catalytically active enzyme molecules

Pepsin operates at _____ pH by using its ________ carboxyl groups for catalysis ("Aspartic Proteinase")

low, aspartic acid

Intracellular Protein Turnover

Turnover rate depends on metabolic state .e.g., greater protein degradation, when nitrogen intake is low, because cells need amino acids to make vitally essential proteins

Two Major Pathways for Intracellular Protein Turnover

Lysosomal/Phagolysosomal Pathway

Ubiquitin-dependentPathway

Lysosomal/Phagolysosomal Pathway

Lysosome is an acidic compartment, where proteins undergo isoelectric expansion (partial unfolding).

Low pH makes them more susceptible to proteolysis

Ubiquitin-dependent Pathway

Ubiquitin is a 8.5-kDa protein that is enzymatically joined to poorly folded proteins.

Ubiquitinated proteins are degraded in proteasomes, barrel-like macromolecular protease complexes

AA & Na+are transported togetherin same direction....called a ____________

symporter

Transport driven by _____________: ______ Na+ in intestinal lumen _____ Na+in brush border cell

trans-membrane ion gradient. High ,Low

Cell transporters use ______________ to drive transport(drives Na+out &K+in)

ATP hydrolysis

Amino acid transporters maintain _______________

chemiosmotic Na+concentration gradient

Nitrogen Balance

Relates Nitrogen intake to Nitrogen excretion

True nitrogen balance:

Intake = Excretion

Positive nitrogen balance:

Intake > Excretion

Negative nitrogen balance:

Excretion > Intake

Positive Nitrogen balanceis required for: (4)

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

Negative Nitrogen balance occurs during: (3)

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

Marasmus

Grossly underweight
No body fat
Muscle wasting
Old man's face
Normal hair
Lethargic

Malnutrition associated with ____________________________________, with little/no edema.

extensive tissue and muscle wasting

Features of marasmus (2)

loose folds of skinhanging over buttocks

Severe deficiency of nearly all nutrients, especially protein, carbohydrates, and lipids

"_______________" resulting from inadequate intake of Protein & Calories (marasmus)

Protein-Energy Malfunction

Kwashiorkor translation

Translation: "sickness last baby getsthe new baby arrives"

Kwashiorkor:

Acute childhood protein malnutrition

How does Kwashiorkor differ from marasmus?

Inadequate protein intake, BUT otherwise adequate caloric intake

Characteristics of Kwashiorkor (3)

Irritability (neurotransmitter deficit)

Enlarged liver(fatty infiltrates)

Abdominal edema-caused by hypoalbuminemia

Transaminases Use Vitamin _____ Coenzymes

B6

pyridoxal phosphate (PLP)

The active coenzyme form of vitamin B6

Has an aldehyde

Pyridoxamine phosphate (PMP)

Has CH2-NH2 group

Transaminases catalyze two half-reactions:

Amino Acid1+ Enz-PLPα>>>>>a-Keto Acid1+ Enz-PMP

α-Keto Acid2+ Enz-PMP>>>>>>Amino Acid2+ Enz-PLP

Sum: amino acid1+ α-Keto Acid2>>>>amino acid2+ α-Keto Acid1

Mechanism of Enzyme-bound Reactions (2 half-reactions)

1st Half-reaction:
-Aldimine forms
-Converts to ketimine
-Hydrolyzes to ketoacid

2nd Half-reaction
is reverse of 1st half-reaction, using R2-KA to make R2-AA

Aldimine

H-C=N-CH-R1

Ketimine

H2-C-N=C-R1

_____ deficiency impairs many AA reactions and formation of some AA-derived neurotransmitters

B6

________ bunds extremely tightly in transamination

PLP

Transamination reactions are fully ________

reversible

Transamination reactions Keq

Keq= 1 (same bonding in substrates and products).

Alanine:α-Ketoglutarate Transaminase

Alanine + α-Ketoglutarate > Glutamate + Pyruvate

NH3 from alanine and carboxyl from α-Ketoglutarate is switched to form new products

Glutamate:Oxaloacetate Transaminase

Glutamate + Oxaloacetate > α-Ketoglutarate + Aspartate

NH3 from glutamate and carboxyl from oxaloacetate is switched to form new products

Transamination is ________

selective

All but _____ amino acidsare transaminated

4

_________ & __________ are secondary amines, and, as such, they lack primary amino groups needed to undergo transamination

proline and hydroxyproline

If ______ were to undergo transamination, the keto acid would cyclize to form a toxic nonmetabolite

lysine

N of NH2 attacks carbonyl

If ______ were to undergo transamination, the keto acid would dimerize into a toxic nonmetabolite

threonine

Transaminases ________ bind lysine or threonine

CANNOT

3 possible routes for deamination

oxidative, hydrolytic, eliminative

Glutamate Dehydrogenase (2) and reaction

Major route for oxidative deamination

Regenerates the amino acceptor (α-ketoglutarate) and provides ammonia, either for re-utilization ordisposal as urea

glutamate + NAD+ + H2O > α-ketoglutarate + NADH + NH3

GDH is located in the

mitochondrial matrix

Draw the structure of α-ketoglutarate

Slide 29

Glutamate Dehydrogenase Mechanism

1st half-reaction oxidizes the amine to a protonated imine, with reduction (hydride transfer) to form NADH

2nd half-reaction hydrolyzes the imine to a keto acid

Sum of Both Reactions:
Glu + NAD+ + imine + H2O > aKG + Imine + NADH + NH3

Glu + NAD+ + H2O >> aKG + NADH + NH3

NAD+ > NADH

Gains H- (reduced)

NADH > NAD+

Lose H- (oxidation)

By storing electrons, ________ & _______ allow cells to manage chemical energy. Also, they are natures batteries

NADH & NADPH

______________ yields energy needed to make 3 mol ATP (Process called:__________________)

NADH oxidation yields energy needed to make 3 mol ATP (Process called: Oxidative Phosphorylation)

Structural differences between NAD+ and NADP+

NAD+ is oxidized

NADP+ has another phosphate group

The part that undergoes redox is a Nitrogen benzene with amide attached (not proper name)

GDH uses _____ or _____

NAD+or NADPH

______ is used in the oxidative deamination reaction

NAD+ (i.e.formation of ammonia and α-ketoglutarate)

The coupling of _______ with __________ allows foroxidative degradation of 14 other amino acids.

What are the exceptions?

GDH, transaminases

Exceptions: Pro, Hyp, Thr, Lys, & His

NADH

is re-oxidized to NAD+ in Oxidative Phosphorylation

α-KA

enters Tricaroxylic Acid Cycle (Citric Acid Cycle)

Excess NH4
+

enters the urea cycle

Operating in the opposite direction ,_____ drives reductive amination to make glutamate

How?

NADPH

hydrolysis? (check)