Biochemistry 2 Flashcards
the 6 enzyme classes (and their functions)
1) oxidoreductases: redox reactions
2) transferases: transfer of chemical group
3) hydrolase: lysis by water
4) lyase: cleavage reaction not using water
5) isomerase: change of molecular conformation
6) ligase: joining of 2 compounds
serine protease mechanism
Substrate binds.
Ser-195 attacks (Ser is very reactive due to His and Asp).
Transition state is stabilized.
Peptide bond is cleaved via hydrolysis.
active site specificity of chymotrypsin
bulky, hydrophobic residues
active site specificity of trypsin
positively charged residues (Arg, Lys)
active site specificity of elastase
small AAs prevalent in elastin (Gly, Ala, Val)
spontaneous reaction (in terms of Gibb’s free energy)
spontaneous if delta G < 0 (negative)
delta G and Keq at equilibrium
delta G = 0
Keq = Q
biochemical reactions vs chemical reactions
Reactions in the body are never at equilibrium.
Some processes are solid phase reactions (not in solution).
removal of product drives the reaction _________
forward
enzymes/catalysts
Stabilize the transition state (lower transition state energy).
Do NOT change delta G or Keq.
catabolism
Breakdown.
Burn fuel for storage or ATP use.
anabolism
Build-up.
Burn ATP for biosynthetic purposes, active transport, mechanical work.
velocity
The amount of product formed per unit time.
Initial velocity is equal to the linear part of the curve.
1/2 Vmax
where Km = [S]
Km
Affinity for a substrate.
Larger Km = weaker affinity.
Never changes.
Always positive.
how to measure enzymatic activity in a sample
Michaelis-Menten.
Use saturating amounts of substrate (»>Km).
how to measure substrate levels
Michaelis-Menten.
Use low substrate levels with respect to Km.
Kcat
Measures the catalytic power of an enzyme.
Kcat = Vmax/[enzyme]
Vmax
Maximal activity for a sample.
More enzyme causes a higher Vmax (up to a point).
Lineweaver-Burke Plot
1/v = (Km/Vmax) (1/S) + (1/Vmax)
slope: Km/Vmax
x-intercept: -1/Km
y-intercept: 1/Vmax
deficiency of enzyme activity: causes
Lack of enzyme.
Defective enzyme.
Lack of substrate/cofactor.
enzyme regulation by location
Enzymes are only expressed in certain tissues.
Ex: ALT (alanine transaminase): internal liver enzyme; high levels = lots of damage
Ex: alpha-1 antitrypsin: indicator of liver damage; secreted by liver and taken up by lungs
zymogen
Inactive form of the enzyme.
Proteolytic cleavage rapidly opens up the active site.
Ex: prothrombin/thrombin and fibrinogen/fibrin in blood clotting cascade.
blood clotting cascade
Damage/trauma activates an enzyme.
Cascade of proteins (1 or 2 trigger, millions activated).
Prothrombin is soluble.
Gamma-carboxylation is added to glutamates on prothrombin (vit K dependent).
Gamma-carboxylation binds Ca2+, so prothrombin can bind to the membrane.
Prothrombin is cleaved to thrombin, an active serine protease, on the membrane.
Fibrinogen is cleaved to fibrin.
Fibrin forms cross-linnked clot.
warfarin/coumadin
Vitamin K analogue.
Interferes with gamma-carboxylation of prothrombin.
Reduces over-clotting in patients that clot too readily.
heparin
Short-acting anticoagulant.
Promotes antithrombin-thrombin complex formation.
Antithrombin is an inhibitor that binds tightly to thrombin,
Once bound together, the complex is degraded, so it reduces the thrombin available for clotting.
Reduces clotting.
elastase inhibition
Elastase is released by lung neutrophils to neutralize foreign particles.
Alpha1-antitrypsin/alpha1-antiprotease inhibit elastase normally.
If alpha1-antitrypsin is defective, then unregulated elastase activity destroys elastin, causing scarring and emphysema.
Defective alpha1-antitrypsin caused by smoking (oxidation of sulfur to sulfoxide).
Treatment: intravenous alpha1-antitrypsin.
phosphorylation of protein
Phosphorylation/dephosphorylation modifies the charge of an AA residue.
Changes structure/function/activity of enzyme.
competitive inhibitors
Interact with binding site.
Same Vmax.
Increased Km (decreased affinity).
noncompetitive inhibitors
Do not interfere with substrate binding.
Decreased Vmax.
Same Km.
irreversible inhibitors
Type of non-competitive inhibitor.
Modifies the enzymatic AAs.
Decreases Vmax.
Same Km.
Ex: DIFP inhibits serine proteases
allosteric affectors
Inhibit or activate.
Usually multiple subunits (cooperativity).
Have R and T forms.
Do not follow Michaelis-Menten kinetics.
Often regulate a reaction pathway.
Highly regulatable by substrate concentration.
Ex: PFK-1: AMP activates, ATP inhibits
product inhibition
The immediate product of an enzyme binds to the enzyme and inhibits its activity.
Ex: hexokinase: G6P inhibits
feedback inhibition
The product inhibits an earlier step of the reaction.
Retinoblastoma
Rb+, Rb+ is normal
Loss of one Rb+ –> predisposed to develop tumor.
Loss of two Rb+ –> induces tumor formation.
E2F needed for transcription of genes needed for cell growth.
E2F is regulated by pRB.
Phosphorylated pRb is inactive which allows E2F to transcribe genes for S phase growth.
Phosphorylation state of pRb is regulated by cdks (cyclin D or E).
Cell growth is accelerated if pRb is lost.
p53
p53 is stabilized by phosphorylation that occurs during stress.
When DNA is damaged, p53 induces transcription of p21 gene.
p21 binds to cdk/cyclin complex and halts the cycle.
p21 binds to PCNA and inhibits replication fork.
p53 halts the cell cycle and prevents apoptosis.
Without p53, p21 is not induced, the cell cycle is not halted, and cells will replicate damaged DNA.
Rb gene suppresses
retinoblastoma
p53 gene suppresses
sarcomas, carcinomas
NFC-1 gene suppresses
neuroblastoma
APC gene suppresses
colon, stomach
BRCA gene suppresses
breast cancer
oncogenes
Activate cell division in response to growth factor stimulation.
Dominant effect: only 1 gene needs to be altered.
Signal Transduction
Signals from outside the cell affects gene expression.
Receptors penetrate cell membrane and have enzymatic activity.
Phosphorylation of Tyr residues allow interactions with other members of cascade.
Tumor cells can generate their own growth signals.
altered growth factors
Simian Sarcoma (sis) oncogene encodes PDGF molecule and can induce signal transduction.
altered growth factor receptors
Mutant forms stimulate growth even in absence of growth factor.
Ex: epidermal growth factor receptor
Family members: ErbB, HER2
Ras signalling
Ras is active when bound to GTP, and inactive when bound to GDP (switches between states by GAP).
Causes signal transduction to nucleus.
Mutant Ras is locked “on” –> excessive signal transduction.
NF1 gene
Encodes neurofibromin protein.
Neurofibromin contains a GAP domain, possibly acts through Ras.
Neurofibromatosis: benign neurofibromas on skin due to defective signal transduction.
c-Fos and c-Jun
Transcription factors.
Bind to AP1 sites.
Too much of these cause continuous growth due to too much signal transduction.
Myc gene
Myc is a transcription factor that regulates 15% of all genes (~3,000 genes).
Myc binds to enhancer sequence and recruits HATs.
Mutant Myc upregulates genes involved in cell proliferation.
Burkitt’s Lymphoma
Translocation involving chromosome 8 (encodes Myc gene) .
When translocated, Myc is constituitively expressed.
Leads to leukemia and lymphomas.
HPV (and other DNA viruses)
T-antigen sequesters Rb and p53.
Takes “brakes” off the cell cycle.
E7 targets Rb.
E6 targets p53
characteristics of cancer
Evade apoptosis. Self-sufficiency in growth signals. Insensitive to anti-growth signals. Limitless replication potential (increased telomerase activity). Angiogenesis. Tissue invasion. Metastasis.
Familial Adenomatous Polyposis Coli (APC)
Loss of APC causes colon cancer.
maltose
glucose + glucose
alpha 1,4 linkage
lactose
glucose + galactose
beta 1,4 linkage
sucrose
glucose + fructose
amylose
Linear.
Polyglucose.
alpha 1,4 linkages.
amylopectin
Branched.
Polyglucose.
Mostly alpha 1,4 linkages.
Some alpha 1,6 linkages to create branches.
cellulose
Polyglucose.
Linear.
Beta 1,4 linkages.
Humans cannot digest.
glycosaminoglycans (GAGs)
Long: 500+ sugars. Linear. Repeating disaccharides. Highly negative (carboxylates, sulfates). Highly hydrated.
major GAGs
Chondroitin sulfate: bone, cartilage, cornea formation.
Keratan sulfate: cornea, CT.
Dermatan sulfate: binds LDL to plasma walls.
Heparan sulfate: aortic wall, basement membrane.
Heparin: anticoagulant.
Hyaluronic acid: cell migration, lubricant
conjugated sugars
Gangliosides: lipids added to sugars.
Glucouronic acid + sugar: more soluble, easier to excrete.
Require activation of sugar by UTP.
glycation of proteins
Occurs without enzyme or cofactor.
Formation of A1C in the blood gives “history” of glucose levels in blood.
glycoproteins
Produced enzymatically.
Secreted or have exterior-facing domains (exception is O-Glc-Nac.
O-linked glycoproteins
On Ser/Thr.
Adds 1 residue at a time.
N-linked glycoproteins
On Asn.
Added in a 14 sugar block.
Constructed on a dolichol phosphate lipid.
Processing occurs during trafficking from ER to Golgi.
Hunter Disease
Congenital disease of glycosylation.
Lack of iduronate sulfatase.
Pompe Disease
Lack of acid a glucosidase.
Target for gene therapy.
Congenital disease of glycosylation.
I-cell Disease
Failure of mannose-6-phosphate trafficking system.
Multi-enzyme disease.
Congenital disease of glycosylation.
mucins
Main component of mucus.
Line/protect epithelial surfaces.
Glycoprotein.
proteoglycans
Core protein O-linked to glycosaminoglycan.
Aggregate on hyaluronic acid.
Hydrated –> provides cushioning.
Charged –> can bind GFs, cytokines, chemokines.
Found in cartilage, dentin, predentin.
glycosylation in biological recognition
Protein recognition of carbohydrate structures occurs via lectin domains.
Differentiate ABO blood groups.
Allows viruses/bacteria to infect.
Mediate cell-cell contact.
importance of glycosylation
Assist in protein folding in ER. Regulate activity. Intracellular transport (mannose-6-P). Regulate half-life of serum proteins. First defense in innate immunity.
Selectin-Carbohydrate interactions
Make contact to slow leukocytes to a roll.
Controls leukocyte trafficking.
calories from lipids
9 kcal/g
calories from proteins
4 kcal/g
calories from carbohydrates
4 kcal/g
digestion of polysaccharides
Mouth: alpha amylase.
Small intestine: pancreatic amylase, brush border disaccharideases (lactase, sucrose-isomaltase).
Colon: bacteria digest unabsorbed carbs.
lactose intolerance
Failure to digest lactose.
Common in African/Asian descent.
Increases with age.
Can be caused by illness that injures the mucosa.
brush border - simple diffusion
Simple diffusion down gradient.
Only “rare” sugars.
brush border - facilitated diffusion
Increases rate of transport down a gradient.
Most common.
Responsive to insulin – increases the number of GLUT4 receptors on the membrane of skeletal muscle, fat, WBC.
Not responsive to insulin – all other transporters, in RBC, brain, pancreas, intestine, brain, kidney.
brush border – active transport
Increases transport rates even against a gradient.
Needed in order to get Na+ out of the cell.
SGLT (sodium linked) indirectly uses active transport.
Glucose and Na+ enters the cell through SGLT-1.
Glucose exits the cell into blood bia facilitated diffusion through GLUT-2.
ATP is needed to pump Na+ out of cell.
active transport of glucose in kidney
Prevents loss of glucose to urine.
factors affecting glycemic index
Sugar content (glucose high, fructose low).
Type of starch (amylose low, amylopectin high).
Physical barriers (bran low).
Viscosity of soluble fiber (apple).
Fat and protein content (affects gastric transport).
Acid content (affects gastric transport).
Food processing (rolled oats low, quick oats high).
Cooking (al dente high).
complete glycolysis reaction
glucose + 2 Pi + 2ADP –> 2 pyruvate + 2ATP + 2NADPH + 2H+ + 2 H2O
glycolysis net reaction
glucose + 2ADP + 2Pi –> 2 lactate + 2ATP + 2 H2O
red blood cells
Lack mitochondria.
Depend on glycolysis for ATP production.
Produce lactate to regenerate NAD+.
Most common glycolytic disorder in RBCs
Pyruvate kinase deficiency.
Causes persistent anemia.
Tarui’s Disease
Mutations in PFK-1.
Causes exercise intolerance.
abnormal pyruvate kinase
Four isozymes: M1 (muscle), M2 (muscle), L (liver), R (RBC).
Defect in a PK causes a disorder in that tissue.
hexokinase
Can phosphorylate other hexoses.
Ubiquitous (in many tissues).
Lower Km (higher affinity) for glucose.
Inhibited by G6P.
Glucokinase
Can only phosphorylate glucose.
Only in liver and pancreatic B cells.
Higher Km (lower affinity) for glucose.
NOT inhibited by G6P.
Takes glucose out of circulation.
MODY
Mature Onset Diabetes in the Young.
NOT associated with obesity or high blood lipid levels.
Pancreatic B cells do produce insulin.
Associated with mutations in glucokinase gene, or genes for TFs that regulate transcription of liver/pancreatic B cell genes.
Without glucokinase, the amount of ATP produced in response to elevated blood sugar is reduced, causing less insulin secretion.
glyceraldehyde-3-phosphate DH
Uses phosphate and NAD+.
Negative cooperativity.
Less sensitive to changes in substrate concentration.
Much lower Km.
NAD+
Required to make GA3P (and DHAP). Must be recycled to continue glycolysis. NAD+>>>NADH. Precursor to niacin. Intermediate to tryptophan synthesis. Deficiencies cause D3 (dematitis, diarrhea, dementia).
pellagra
Niacin/NAD+ deficiency. Places with corn. Causes D3 (dermatitis, diarrhea, dementia) and death.
glucose-6-phosphate can also be used for
glycogen, polysaccharides, glycoproteins, ribose, NADPH
fructose-6-phosphate can also be used for
ribose, pentoses
glyceraldehyde-3-phosphate can also be used for
ribose, pentoses
dehydroxyacetone phosphate can also be used for
fat/phospholipid metabolism
3-phosphoglyceride can also be used for
serine
ACoA can also be used for
FAs, cholesterol, steroid hormones, oxidative metabolism
2-deoxyglucose
Used for PET imaging.
Targets hexokinase.
arsenate
Targets GA3P DH.
Substitutes for phosphate.
flouride
Targets enolase.
PET imaging
Shows hotspots for glycolysis.
Can see if metabolic activity in tumor is knocked out.
Detects metastasis.
% ethanol absorption in stomach? intestine?
20% stomach
80% intestine
Eating food slows gastric emptying, thus slows absorption (esp. fatty foods)
factors affecting elimination rate of ethanol
Sex (male > female).
Body composition (fat > lean).
Tolerance (experience > novice).
Alleles (ADH).
NAD/NADH levels in alcohol metabolism
Alcohol clearance produces lots of NADH.
High NADH inhibits gluconeogenesis, promotes triglyceride synthesis.
Acetaldehyde increases TFs driving FA synthesis.
MEOS
Microsomal Ethanol Oxidizing System. Activated with chronic alcohol consumption. Cytochrome p450 based. Smooth ER. 2/3 of total ethanol oxidation. Affects drug metabolism. Inducible.
ADH1B*2
Polymorphism of ADH1.
Common in asian descent.
Ethanol is metabolized more quickly, less of an effect.
ADH2*2
Polymorphism of ADH2.
Common in asian descent.
Converts acetaldhyde to acetic acid, but is slow.
Acetaldehyde causes facial flushing, high HR.
Uncomfortable.
Disulfiram/Antabuse
Inhibits ADH2.
Creates an unpleasant reaction to alcohol.
FDA-approved for treatment of alcohol use disorder.
thiamine deficiency
Common in alcoholics (poor diet, absorption).
Wernicke’s Encephalopathy.
Korsakoff’s Psychosis.
Wernicke’s Encephalopathy
Thiamine deficiency. C - confusion O - opthalmoplegia A - ataxia T - thiamine treatment
Korsakoff’s Psychosis
Thiamin deficiency. R - retrograde amnesia A - anterograde amnesia C - confabulation K - Korsakoff's psychosis
metabolism of ethylene, methanol
Metabolized by same enzymes as ethanol.
Produce toxins (cause death, blindness, kidney failure).
Treatment: give ethanol.
Drug: Fomepizole (inhibits ADH, not v effective).
thiamine cofactor
Used in PDH and Isocitrate DH.
Vitamin B1.
Does carboxylations and 2 carbon transfers.
Deficiency leads to beri-beri and Wernicke’s.
lipoate cofactor
Used in PDH and isocitrate DH.
Activates acetate.
Target of arsenic.
FAD cofactor (specifically in PDH/isocitrate DH)
Regenerates lipoate so rxn can continue.
Vitamin B2/riboflavin.
riboflavin deficiency
cheilosis
glossitis
beri-beri
Thiamine deficiency.
DRY: peripheral neuropathy, tingling hands/feet, involuntary eye movement.
WET: cardiac issues
PDH complex deficiency (results and therapies)
Metabolic acidosis (build up of lactate, pyruvate, alanine). Neurological disorders ( cerebellar dysfunction).
Therapies: Cofactor supplementation (maximize enzyme activity). Ketogenic diet (reduce pyruvate load). Bicarbonate (control acidosis). Dichloroacetate (reduce PDH inhibition, to treat acidosis).
metabolic disease associated with succinate DH
phaeochromocytoma,
paraganglioma
metabolic disease associated with fumarase
leiomyomas
metabolic disease associated with isocitrate DH
tumors (gliomas, AML)
transketolase
transfers 2 carbons using thiamine
transaldolase
transfers 3 carbons
rate-limiting step of PPP
1st step (G6P –> phosphogluconolactone + NADPH).
Activated by insulin.
Allosterically inhibited by NADPH.
Saturated with inhibitor, starved for substrate (until it is needed).
reactions that use NADPH
Synthesis of: FA, cholesterol, neurotransmitters, nucleotides.
Detoxification of: oxidized glutathione, cytochrome p450.
NADPH vs NADH
Same function, different structure.
Allows enzymes to recognize structures, and keep ratios opposite for different reactions.
myeloperoxidase
Can produce hypochlorous acid (bleach).
Kills bacteria and fungi.
Neutrophils.
Chronic Granulomatous Disease (CGD)
Defect in NADPH oxidase.
Cannot form myeloperoxidase to kill bacteria.
antioxidants
Get rid of free radicals.
Accept electron and decay without further damage.
Vitamins A, C, E.
reduction of free radicals
Superoxide dismutase converts superoxide to hydrogen peroxide.
Glutathione peroxidase reduces peroxides using NADPH.
effects of hypoglycemia
ADRENERGIC (autonomic):
Trembling, palpitations, sweating, anxiety, nausea, hunger, tingling.
NEUROGLYCOPENIC:
Headache, confusion, weakness, drowsiness, vision changes, difficulty speaking, dizziness, tiredness.
gluconeogenesis locations
Mainly in cytoplasm.
Pyruvate carboxylase is in mitochondria.
G6P’ase in ER.
90% liver
10% kidney
Cori cycle
Lactate from RBC/muscle is shuttled thru blood to liver.
Gluconeogenesis converts lactate to glucose.
Glucose is returned to RBC/muscle for energy.
Spend 6 ATP, gain 2 ATP.
Cahill Cycle
Alanine Cycle.
Pyruvate converted to alanine.
Alanine travels thru blood to liver.
Gluconeogenesis converts alanine to glucose.
Spend 6 ATP in liver, get 2 ATP in muscle/RBC.
when is glycogen used as a main source of glucose?
4-16 hours after eating
Faconi-Bickel Syndrome
GLUT2 deficiency.
Glucose transport is blocked.
Limited glucose uptake causes hypoglycemia.
Cannot export glucose from glycogen, so buildup of glycogen.
limiting step of glycogen synthesis
Glycogen synthase.
Adds UDP-glucose 1 residue at a time.
glycogen primer
Glycogenin.
Self-glycosylates.
Adds UDG-G to Tyr-194.
branching enzyme
Transfers a block of residues from a terminal end.
Creates a 1-6 branch.
Must be 4+ residues away from an existing branch.
debranching enzyme
Transfers 3 residues of a branching chain to a different chain, leaving 1 branched residue.
Cleaves remaining 1 residue via hydrolysis.
Type I / von Gierke (glycogen storage disease)
G6P’ase deficiency (a).
Or defect in transport of G6P to ER (b).
Severe fasting hypoglycemia.
Unresponsive to glucagon, epinephrine.
Increased liver size due to excess glycogen.
Decreased gluconeogenesis causes lactic acidosis.
Treatment: cornstarch
Type II / Pompe (glycogen storage disease)
Lysosomal acid maltase deficiency.
Inability to digest polysaccharides causes cell inclusions.
A type of LSD.
Type III / Cori (glycogen storage disease)
Debranching enzyme deficiency.
Abnormal glycogen with short outer branches.
Growth retardation, hepatomegaly, hypoglycemia.
Not as severe as Type I bc gluconeogenesis still possible.
Type IV / Anderson (glycogen storage disease)
Branching enzyme deficiency.
Abnormal glycogen with few branches.
Weird, long glycogen treated as a foreign body.
Death.
Type V / McArdle (glycogen storage disease)
Muscle phosphorylase deficiency.
Muscle cramps during hard exercise.
No rise in lactate after exercise.
Myoglobinuria.
Type VI / Hers (glycogen storage disease)
Liver phosphorylase or phosphorylase kinase deficiency.
Hypoglycemia.
Gluconeogenesis still possible, so not as severe.
Type VII / Tauri (glycogen storage disease)
Muscle PFK deficiency.
Muscle glycogen content is increased.
Muscle cramps and exercise intolerance.
Helped by fructose (bypasses PFK step).
