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164 Cards in this Set
- Front
- Back
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Ligand |
a molecule bound reversibly by a protein |
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binding site |
where a ligand binds |
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induced fit |
structural adaptation of a protein to bind a ligand |
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heme |
protein-bound ring that contains an iron atom at the center to bind oxygen (porphyrin ring) |
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Fe 2+ |
binds oxygen reversibly |
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Fe 3+ |
does not bind oxygen |
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Prevention of Fe oxidation |
one of open binding sites is occupied by a side chain nitrogen of a His residue |
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globins |
family of oxygen binding proteins |
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myoglobin |
single polypeptide made of 8 a-helix residues with a single heme molecule (bound at His93) |
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equilibrium constant (Ka) |
[PL]/[P][L] - describes affinity of ligand for a protein |
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higher Ka value |
higher affinity of protein for ligand |
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lower Ka value |
lower affinity of protein for ligand |
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theta |
binding sites occupied/total binding sites |
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when theta = .5 |
[L] = 1/Ka |
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dissociation constant (Kd) |
the equilibrium constant for the release of the ligand |
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the more tightly a protein binds to ligand |
the lower the concentration of ligand required to for half the binding sites to be occupied (lower Kd) |
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CO binding |
linear (20000X better than oxygen) |
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Oxygen binding |
at an angle (increases steric strain) |
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myoglobin flexibility |
allows oxygen to pass through and contact heme |
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erythrocytes |
red blood cells |
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red blood cell survival |
120 days |
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RBCs in arteries |
96% saturated with oxygen |
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RBC's in veins |
64% saturated with oxygen |
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hemoglobin |
spherical with 4 heme prosthetic groups (2 a-chain 2 B-chain) |
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a1B1 and a2B2 interface |
strongest because of hydrophobic interactions |
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2 major hemoglobin concentrations |
R and T |
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R state |
oxygen has a higher rate of affinity for binding, low CO2 and H+ affinity, in the lungs |
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T state |
low rate of oxygen affinity, high affinity for CO2 and H+, in the tissues |
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hemoglobin conformational change |
aB subunit pairs slide past each other, narrowing the pocket between B subunits (His HC3 residues of B subunits move close to each other), causing a more planar conformation |
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T --> R transition (lungs) |
oxygen binding, CO2 and H+ releasing |
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R --> T transition (tissues) |
oxygen releasing, CO2 and H+ binding |
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myoglobin binding curve |
hyperbolic |
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hemoglobin binding curve |
sigmoidal |
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homotropic |
ligand and modulator are identical |
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heterotropic |
ligand and modulator are different molecules |
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increased oxygen binding in hemoglobin |
increases oxygen affinity |
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cooperative hill plot |
nh (slope) > 1 |
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negatively cooperative hill plot |
nh (slope) < 1 |
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hill plot |
plot of logpO2 vs log(theta/1 - theta) |
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tissue pH |
low pH, shifts binding curve to the right |
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lung pH |
high pH, shifts binding curve to the left |
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hemoglobin protonation |
His146 protonation forms an ion pair with Asp94 to stabilize T state ( why H+ binding releases oxygen) |
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CO2 binding |
binds at amino terminal of a-subunits |
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2,3 bisphosphoglycerate (BPG) |
- decreases the affinity of hemoglobin for oxygen - bound by positive amino acids in the B cavity, stabilizing the T-state - increase shifts binding curve to the left |
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high altitudes |
BPG increases (does not affect oxygen binding in lungs) but does decrease oxygen affinity in tissues, allowing for the release of oxygen to increase |
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sickle cell anemia |
Hemoglobin S has Glu --> Val which makes negative charge --> neutral creating hydrophobic pockets. These pockets cause the glucose molecules to aggregate into tubular fibers, causing the sickle shape |
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Phi bond angle |
N - Ca bond rotation
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Psi bond angle |
Ca - C bond rotation |
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a-helix |
spiral of amino acids (3.6 [5.4 A] residues per turn) with R groups on the outside - long combos of negative or positive charge prevent a-helix from forming because of electrostatic repulsions - positive and negative amion acids often 3 away from each other to form stabilizing ion pairs |
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B - sheet |
sheet of amino acids with R groups above and below plane of sheet |
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antiparallel B-sheet |
more stable due to linear H-bonding |
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parallel B-sheet |
less stable due to H bonding at an angle |
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B-turn |
180 degree turn made by 4 amino acid residues to connect strands of antiparallel B-sheet |
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hydrophobic interactions |
proteins fold to bury hydrophobic interactions |
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B-sheet stabiliziation |
more stable when twisted to the right |
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aB barrel |
each parallel B segment is attached to its neighbor by an a-helix segment |
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denaturation |
a loss of 3D structure sufficient to cause loss of function |
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heat denaturation |
destroys hydrogen bonds |
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urea denaturation |
destroys hydrogen bonds |
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Anfinsen experiment |
Ribonuclease A is denatured by urea but when urea removed it spontaneously refolded, proving that amino acid sequence is responsible for tertiary structure |
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Hsp70 |
helps proteins refold by binding to hydrophobic residues (chaperonins also do this) |
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Crude extract |
the proteins solution obtained from lysing a cell |
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fractionation |
separating proteins into groups of different sizes |
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dialysis |
use of a selectively permeable membrane to remove small solutes |
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ion-exchange chromatography |
solid matrix uses positively charged groups to bind proteins at different pH |
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cation-exchange chromatography |
solid matrix uses negatively charged groups to bind proteins at different pH |
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size exclusion chromatography |
large proteins cant enter matrix cavities and elute faster |
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affinity chromatography |
targets proteins with a specific ligand to bind only that protein |
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electrophoresis |
proteins migrate from negative to positive and are slowed by polyacrilimide gel (larger ones remain towards top, smaller towards bottom) |
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SDS |
gives all proteins negative charge (anode (+) is at bottom, cathode (-) is at top) |
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2D electrophoresis |
pH gradient separates proteins at top by pI, then current seperates by size |
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activity |
total units of enzyme in a solution |
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specific activity |
number of enzymes per unit miligram of total protein |
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Enzyme |
molecule that helps lower the activation energy of a reaction |
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cofactor |
additional chemical component that an enzyme requires (Mg 2+, Fe 2+, Mn 2+, Zn 2+) |
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coenzyme |
complex organometallic molecule requried by some enzymes |
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prosthetic group |
coenzyme tightly or covalently bound to an enzyme |
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holoenzyme |
enzyme with cofactor/coenzyme bound (active) |
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apoenzyme/apoprotein |
enzyme without cofactors/coenzyme (inactive) |
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enzyme naming |
"substrate + ase" |
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oxidorecutases |
transfer of electrons (hydride ions or H atoms) to reduce or oxidize |
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transferases |
group transfer reactions |
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hydrolases |
hydrolysis reactions (transfer of functional groups to water) |
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lysases |
cleavage of C-C, C-O, or C-N bonds by elimination, leaving double bonds or rings, or additon of groups to double bonds |
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isomerases |
transfer of groups within molecules to yield isomeric forms |
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ligases |
formation of C-C, C-S, C-O, and C-N bonds condensation reactions coupled to cleavage of ATP or similar cofactor |
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Kinases |
transfer of phosphate groups |
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Initial rate (Vo) |
in beginning of reaction, [S] can be treated as constant so Vo is a function of [S] - at low concentrations, Vo increases linearly with [S], but this plateaus at high [S] |
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rate limiting reaction |
conversion of [ES] to E + P |
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enzyme saturation |
all [E] pushed to [ES] so increasing [S] has no effect on rate |
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steady state kinetics |
[ES] remains approximately constant because the rate of formation of [ES] is the same of its rate of breakdown (to product and back to substrate) |
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Km |
Micahelis Constant, tends to be similar to cellular concentrations of its substrate |
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bisubstrate reactions |
enzyme binds 2 or more substrate molecules |
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tenary complex |
both molecules must bind before forming products (intersecting lines with varing [S]) |
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ping pong complex |
molecules can bind independent of each other (parallel lines with varing [S]) |
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Competitive inhibitor (reversible) |
competes with substrate for active site on enzyme (structurally similar) - does not lower Vmax - a coefficeint |
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uncompetitive inhibitor |
binds at its own site and only to the ES complex - lowers Vmax and Km - a' coefficient |
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mixed inhibitor |
binds at its own site to both E and ES - can lower Vmax and Km (has both a and a' coefficients) |
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noncompetitive inhibition |
when a = a' |
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Irreversible inhibitors |
bind covalently with enzyme, destroy enzyme functional group, or form stable noncovalent interactions with enzyme |
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suicide inhibitors |
undergo first few steps of enzymatic reaction which transforms them into a very reactive, irreversibly bound inhibitor |
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transition state analogs |
bind more tightly to the [ES] complex and lead to inhibition |
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pH and enzyme activity |
can protonate or deprotonate side chains |
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Energy transductions |
changes from one form of energy to another in the cell (or elsewhere) |
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1st law |
energy can neither be created nor destroyed |
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2nd law |
entropy must increase in all natural processes |
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cells and the 2nd law |
living systems are never at equilibrium with their surroundings so constant transactions with their surrounds explain the increase of entropy |
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gibbs free energy (G) |
amount of energy available for doing work - negative (delta)G, exergonic and spontaneous, positive (delta)G, endergonic and nonspontaneous |
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Enthalpy (H) |
reflects the heat content of a reacting system - negative (delta)H, exothermic, positive (delta)H, endothermic |
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Entropy (S) |
randomness or disorder
- positive (delta)S, more disordered, negative (delta)S, more complex |
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buffered aqueous solutions |
pH and [H2O] are constant |
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biochem assumptions |
- cell operates at constant T and P - [H+] = 10^-7 M - [H2O] = 55.5 M - [Mg2+] = 1mM |
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Keq > 1 |
spontaneous (-(delta)G) |
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Keq < 1 |
nonspontanous (+(delta)G) |
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What enzymes can change/not change |
can change activation energy, cannot change equilibrium constants |
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multistep reactons |
free energies are additive |
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coupling reactions |
equilibrium constants coupled by multiplication to allow unfavorable reactions to be incorperated into biological processes |
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(delta)G and ATP |
breaking off of 3rd phosphate from ATP decreases charge repulsion leading to more stable molecule (energy released during ATP hydrolysis is greater than standard free energy) |
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phosphorylation potential |
phosphorylation occurs naturally in the cell in the presence of Mg2+ |
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cellular ATP concentrations |
held far above equilibrium because as [ATP] diminishes so does the phosphorylation potential |
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Energetically favorable phosphorylated compounds |
-Phosphoenolpyruvate (>pyruvate) -1,3 bisphosphoglycerate (>3phosphoglycerate) -phosphocreatine (>creatine) -acetyl Co-A (acetate + CoA) |
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Phosyphorylated products more stable because |
bond strain from electrostatic repulsion is relieved by charge separation |
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products are stabilized by ionization |
ATP, Acetyl Co-A, thioesters |
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products are stabilized by isomerization |
PEP |
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products are stabilized by resonance |
creatine |
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ATP hydrolysis reaction steps |
1. Pi binds to 1 reactant 2. other reactant replaces P to form product |
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High energy compounds |
(delta)G < -25kj/mol |
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low energy compounds |
(delta)G > -25kj/mol |
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ATP energetically stable because |
dont want it to donate phosphate groups without presence of enzyme (like to water or other molecules) |
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y-attack |
yields phosphate group + ADP - transport processes (Na+ and K+ pumps) - skeletal muscle - formation of nucleoside triphosphates |
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B-attack |
yeilds pyrophophyrl + AMP |
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a-attack |
yields adenylyl + 2Pi - energy intensive reactions - DNA and RNA polymerization |
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Ways of forming more ATP |
-adenylate kinase (2ADP > ATP + AMP) - ADP + PEP > ATP - creatine kinase (ADP + PCr > ATP + Cr) - inorgantic phosphate: ADP + (polyP)n+1 > ATP + (polyP)n |
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EMF |
electromotive force that drives electrons between difference in electron affinity between molecules |
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reducing agent |
electron donor (oxidized) |
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oxidizing agent |
electron acceptor (reduced) |
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biological oxidation |
coincides with the loss of hydrogen (dehydrogenation) |
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Methods of Electron Transfer |
1. directly as electrons (metals) 2. as hydrogen atoms 3. as hydride ions 4. direct combination with oxygen |
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Reduciton potential (Eo) |
relative affinity of electron acceptor of each redox pair for electrons |
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electron flow |
from lower E to higher E (larger E will be reduced) |
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(delta)E |
E(reduction) - E(oxidation) |
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biological electron carriers |
NADH and NADPH (catalyzed by oxidoreductases) |
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NADH |
NAD+ to NADH ratio is high, favoring oxidations |
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NADPH |
NADPH to NADP+ ratio is high, favoring reductions |
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flavoproteins |
enzymes that catalyze redox reactions using FMN's or FAD's as coenzymes (light receptors) - flavin ring can be reversibly reduced with 1 or 2 H+ atoms |
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glycolysis |
breakdown of glucose to 2 pyruvate to yeild 2 ATP and 2 NADH |
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pyruvate breakdown |
aerobic conditions: Acetyl CoA anerobic conditions: lactate or ethanol and CO2 |
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glycolysis reactions that require ATP |
glucose > glucose 6-phosphate Fructose 6-phosphate > Fructose 1,6-bisphospate |
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glycolysis reactions that yield ATP |
1,3 bisphosphoglycerate > 3-phosphoglycerate phosphoenolpyruvate > pyruvate |
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glycolysis reactions that yield NADH |
glyceraldehyde 3-phospate > 1,3 bisphosphoglycerate (requires 2Pi) |
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reasons for phosphorylation |
-phosphorylated glycolitic intermediates cannot leave the cell because of a lack of transporters -high energy phosphate compounds donate phosphoryl groups to ADP to form ATP -binding energy from phosphate groups to enzymes lowers activation energy and increases specificity (because of Mg2+) |
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phosphofructokinase inhibition |
when the cell has ample amounts of ATP |
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3-phosphoglycerate > 2-phosphoglycerate |
has 2,3 bisphosphoglycerate as intermediate which is essential for hemoglobin |
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aerobic vs. anerobic |
30 ATP aerobically, 2 ATP anerobically so 15X as much glucose needed without the presence of oxygen |
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glycogen metabolism |
excess glucose is converted to glycogen in animals and starch in plants |
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glycogen particle (B-particle) |
55,000 glucoes molecules |
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a-rosette |
20-40 glycogen B-particles |
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glycogen importance |
the brain cannot burn fatty acids as fuel |
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glycogen phosphorylase |
breaks off glucose molecules through phosphorylation of 1-4 linkage but halts 4 away from 1-6 linkage |
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oligo (1-6) to (1-4) glucantransferase |
debranches the 1-6 linkage |
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glucose 6 phosphate |
enter glycolysis in skeletal muscle |
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the liver |
reduces glucoes 6 phosphate to glucose and Pi |
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Sugar nucleotide uses |
1. sugar nucleotide formation is irreversible (drives reactions forward) 2. nucleotides can interact with enzymes to increase binding energy 3. nucleotide group is excellent leaving group 4. cells tag hexoses with nucleotides to set them aside for reserves (glycogen) |
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amylo (1-4) to (1-6) transglycolosase |
branches glycogen during synthesis |
