Enzymes and Bioenergetics Flashcards
Km
- Km= 1/2 Vmax
- Lower Km …high affinity of enzyme for substrate (need less conc of substrate to saturate)
- Higher Km…lower affinity of enzyme for substrate (need inc conc of substance to saturate)
Vmax
Vmax = velocity at which enzyme is saturated w/ substrate so adding additional enzyme does not inc velocity anymore
Km and Vmax on Lineweaver-Burke Plot
Dbl Reciprocal of M-M —> Lineweaver-Burke Plot (linear)
- Slope = Km/Vmax - Y-int = 1/Vmax - X-int = -1/Km
How do competitive inhibitors affect Lineweaver-Burke plot?
Vmax unchanged but inc Km
So inc/steeper slope and dec x-int (more negative)
How do non-competitive inhibitors affect Lineweaver-Burke plot?
Km unchanged but Vmax dec
So inc/steeper slope and inc y-int (higher)
Competitive Enzyme v Noncompetitive Enzyme
Comp- - Resembles substrate; competes w/ substrate @ ACTIVE SITE; overcome by inc conc of substrate
Non-Comp- Does not resemble substrate; binds to enzyme NOT NEC @ ACTIVE SITE; cannot be overcome by inc conc of substrate
Suicide Substrate (+4 clinical examples)
Irreversible enzyme inhibitor- reacts COVALENTLY on some functional group of enzyme —> poison/inactivate enzyme
Ex) Viagra, Aspirin (modifies cycloxygenase in prostaglandin formation), Flurouracil (inhibits thymidylate synthase in chemo), allopurinol (inhibits xanthine oxidase to treat gout)
3 Characteristics of Reg Enzymes
- Catalyze Irreversible rxns (non-equilibrium)
- Catalyze the rate-limiting step in a pathway (slowest step)
- Catalyze the committed step in a pathway (first step committed solely to formation particular product)
3 Mechanisms of Enzyme Regulation
1- Change amount of enzyme (inc enzyme amount)
**Alt transcription
2-Change catalytic properties of enzyme (inc activity- change kinetics)
**Alt substrate conc, covalent mod, allosterics
3-Multienzyme complexes (inc efficiency)
**Mult enzymes so rxn cont 1 after other w/o mixing product w/ solvent (efficiency)
What is the most important type of reversible covalent modification and what enzymes does it use?
Phosphorylation!
- P added by kinases (transfer from ATP —> hydroxyl on serine/threonine/tyrosine—> phosphate ester linkage)
- P removed by phosphatases (use water to hydrolyze phosphate ester)
How do you know if phosphorylation activates or inactivates?
- If enzyme in CATABOLIC path —> P activates it
- If enzyme in ANABOLIC path —> P deactivates it
Allostery (+ example)
-Allosteric Effector- pos/act or neg/inact effect on enzyme activity by binding to site DISTINCT FROM ACTIVE SITE
- Allosteric act- dec Km, inc Vmax or both
- Allosteric inhib- inc Km, dec Vmax or both
- Usually either end products of pathway or metabolites that reflect energy status of cell
- Acts by changing conformation of enzyme
- Ex) phosphofructokinase-1
- rate limiting step in glycolysis - commits glucose to glycolysis
- allosteric effectors for this enzyme - AMP, ADP, ATP, fructose-2,6-P2 and citrate
- ATP inhibits while ADP and AMP relieve the ATP inhibition
Limited Proteolysis (what is it an example of?)
Irreversible Covalent Modification!
- Cleave peptide bonds at limited # sites
- Remove conformational restraints
- New conformation —> new function —> cascade act
Ex) blood clotting, complement act, etc
Free Energy Change
- Value shows the energy diff b/n substrate and products
- Sign (+/-) shows if the rxn requires/yields energy
+ delta G requires energy (endergonic)
- delta G yields energy (exergonic)
deltaG=0 at equilibrium - *Indep of path by which rxn occurs
- *Rxn goes toward neg deltaG
What is required for coupled enzymatic reactions?
Reactions must share a common intermediate
What is the function of adenine nucleotides?
ATP- major carrier of chemical energy in cell (high energy bonds)
Energy released by hydrolysis of 1 or 2 phosphate bonds —> supplies endergonic rxns
Energy Charge
Index of metabolically available energy in the cell
=[ATP] + 1/2[ADP] /[ ATP] + [ADP] + [AMP]
- If = 1 …all ATP
- If = 0 …all AMP
How does energy charge affect regulation of cell metabolism?
It is used to describe energy state of cell and energy state can regulate enzymes
ATP, ADP and AMP can act as allosteric regulators of many enzymes
Oxidation v Reduction
- Oxid- lose electrons …oxidizing agent accepts electrons
- Red- gain electrons …reducing agent donates electrons
Relationship B/n Change in Free Energy (deltaG) and Diff in Redox Potential (deltaE’)
Pos deltaE’ = Neg deltaG = exergonic and vice versa
2 Major Electron Carriers in Redox
- 1- NADH (transfer hydride ion)
- NAD+ + H+ + 2e- —> NADH
- 2- FADH2 (transfer 2 hydrogen atoms to nitrogen atoms of rings)
- FAD + 2H- + 2e- —> FADH2
What enzymes are involved in detoxification of ROS?
Superoxide Dismutase (for superoxide O2-)
Peroxidase and Catalase (for hydroxide H2O2)
**None for hydroxyl radical b/c reacts so rapidly (*OH)
What are the major components of the respiratory chain?
- Complex I - NADH-linked dehydrogenase
- Complex II - FADH-linked dehydrogenase (succinate dehydrogenase)
- Coenzyme Q - mobile; transfers electrons to cytochromes of complex III
- Complex III- cytochrome c reductase
- Composed of 2 cytochromes (contain heme)
- Cytochrome C - transfers electrons to complex IV
- Complex IV- cytochrome oxidase; transfers electrons to molecular oxygen —> H2O
- Iron sulfur proteins- non-heme iron proteins associated w/ complexes I II III)
- ATP Synthase (Complex V)
How does ETC turn dietary fuel into ATP?
-Fuels –> acetyl CoA –> TCA cycle to become NADH and FADH2 –> enter ETC and created proton gradient –> H+ travels back through ATP synthase and makes ATP
Oxidative Phosphorylation v Substrate-Level Phosphorylation
Oxidative phosphorylation uses oxygen as ultimate electron acceptor and creates proton gradient that provides the energy to produce ATP (**energy comes from H+ gradient)
substrate level phosphorylation in which there is a direct transfer of PO3- from a reactive intermediate to produce ATP (**energy comes from another P bond)
2 Shuttle Systems
- 1- Malate-Aspartate
- Reduce oxaloacetate —> malate in cytosol
- Oxidize malate —> oxaloacetate in matrix
- 2 NADH —> 2 NADH (no energy lost)
- Uses isozymes of malate dehydrogenase
- Mainly in liver and heart
- 2- Glycerol Phosphate
- Reduce DHAP —>glycerol-3-phospahte in cytosol
- Oxidize glycerol-3-phosphate —> DHAP in matrix
- NADH —> FADH2 (lose some energy)
- Uses isozymes of alpha glycerol phosphate dehydrogenase
- Mainly in brain and fast-contracting skeletal muscle
ATP yield from pyruvate dehydrogenase v succinate dehydrogenase
Pyruvate dehydrogenase —> NADH (enters ETC at complex I —> more protons pumped so makes 3 ATP)
Succinate dehydrogenase —> FADH2 (enters ETC at complex II —> less protons pumped so makes only 2 ATP)
Cyanide v dinitrophenol
Dinitrophenol - uncoupler; make the mito membrane more permeable to H+ so dec gradient - dec efficiency and inc heat lost in reaction
Cyanide - site specific inhibitor- bind to specific components of ETC blocking e- transfer (ex- cyanide binds to complex IV)
How is mitochondria related to heat generation in newborns?
Uncoupling protein in brown fat (UCP1 thermogenic) —> 5% efficiency so lots of heat given off; more prevalent in infants (important in warming their blood especially)
How does ATP synthase work?
- H+ gradient/charge separation creates potential energy and H+ wants to go back into matrix but can only do so by entering F0 channel; H+ flow causes stalk to rotate which in turn causes F1 to synthesize ATP
**Protons are pumped out of complexes I II and III
