Biochemistry

exergonic/endergonic

energy exits/enters the system, negative/positive dG

exothermic/endothermic

heat exits/enters the system, negative/positive dH

entropy

dS is always positive, disorder of universe tends to increase

enthalpy

dH = dE + PdV, heat

Gibbs free energy

dG = dH - TdS, negative dG means reaction is spontaneous and favorable, this is determined by both Keq and Q

dG' = - RTlnK'eq

dG = dG' + RTlnQ, Q = Keq but not at any given time

ATP -> ADP + P, dG = -12

activation energy

energy required to produce the transition state, catalyst/enzyme stabilize the transition state and reduce Ea without changing dG

higher Ea means slower reaction rate

drawing a reaction coordinate graph

enzymes

physiological catalysts

increase reaction rate so it happens in a biologically relevant time-frame, not used up in reaction, specific to a reaction (important for regulation)

interact with substrate at active site, always stereospecific and can form specific stereoisomers from non-chiral molecules

can interact with different substrates that have similar chemical linkages

induced-fit model vs. lock-key model

dimers have two similar proteins connected by hydrophobic amino acids or by disulfide bonds

heterodimer- two different proteins

homodimer- two identical proteins

common types:

1. kinases takes phosphate group from donor (ATP)

2. phosphatases removes phosphate group

3. phosphorylases adds phosphate group

3. ligases combine two molecules

4. lyases break apart a molecule, form double bond

5. isomerases convert between isomers

6. transferases transfer functional groups from one molecule to another (sometimes includes kinases and phosphatases)

activating enzymes

zymogen is an inactive enzyme that needs to be cleaved

apoenzyme is an inactive enzyme that needs a cofactor

phosphorylation can activate/deactivate

allosteric interactions can regulate

hydrolyzing enzymes

hydrolysis breaks bonds

lipase- hydrolysis of lipids (triacylglycerol breaks apart into glycerol and 3 fatty acids)

protease- hydrolysis of proteins (proteins are cleaved to activate subunits)

endonuclease- hydrolysis of nucleotides in middle of a strand (restriction enzymes cut at palindromes)

exonuclease- hydrolysis of nucleotides at the ends of a strand

ribonuclease- hydrolysis of RNA (protected from my 5'-caps and 3'-poly A tails)

amylase, glycosidase- hydrolysis of carbohydrates

enzyme regulation

1. regulated at allosteric site

2. regulated by modifications like phosphorylation

on vs. off states

negative feedback- product inhibits enzyme

positive feedback- product activates enzyme

oxytocin is example of positive feedback, needs external regulator to eventually stop process

oxidation/reduction

loss/gain of hydrogen atoms, gain/loss of charge

Bronsted-Lowry acid/base

proton donor/acceptor

Lewis acid/base

electron pair acceptor/donor, usually in coordinate covalent bonds

acid/base-dissociation constant

large Ka/Kb means stronger acid/base

Ka = [H3O+][A-]/[HA]

Kb = [HB+][OH-]/[B]

amphoteric

can act as either acid or base, amino acids

conjugate base of a weak polyprotic acid is always amphoteric

each time a polyprotic acid donates another proton, it becomes a weaker acid

pH

pH = -log[H+], water at 25C has pH = 7

pH + pOH = 14

pKa

pKa = -logKa

lower pKa/pKb is the stronger the acid/base

buffer

weak acid and its conjugate base

bicarbonate buffer system, carbonic acid and bicarbonate

amino acids

memorize their structure, names, letters, properties, physiological pH

Nonpolar: PI GALVY MWF

"my PI goes to Galveston on mon/wed/fri"

Acidic: DE (negative at physiological pH)

Basic: HRK (positive at physiological pH)

alanine - ala - A

glycine - gly - G

valine - val - V

leucine - leu - L

isoleucine - ile - I

proline - pro - P

phenylalanine - phe - F

tryptophan - trp - W

tyrosine - tyr - Y (10.1)

serine - ser - S

threonine - thr - T

cysteine - cys - C (8)

methionine - met - M

lysine - lys - K (10.5)

arginine - arg - R (12.5)

histidine - his - H (6.1)

aspartic acid - asp - D (3.9)

glutamic acid - glu - E (4.1)

asparagine - asn - N

glutamine - gln - Q

amino group (10)

carboxyl group (2)

conservative substitution

binding affinity is not affected by the substitution, indicates that the original amino acid is not involved in binding or does not change conformation of enzyme

if binding affinity goes up or down, it is not conservative

average weight of amino acid

110 Da (g/mol)

Henderson-Hasselbalch equation

pH < pKa, will be protonated

pH > pKa, will be deprotonated

isoelectric point

pH of amino acid where net charge is 0, zwitterion

pI = average of the pKas of the two functional groups

peptide bond

amino group attacks the carboxyl group during synthesis

proteolytic cleavage breaks peptide bonds by hydrolysis

N-C synthesis, N-terminus is synthesized first and written first

disulfide bridge

cysteines are oxidized to cystine in a disulfide bridge

join together multiple subunits of proteins

reducing conditions in a gel will break apart disulfide bridges and thus the subunits

pi stacking

tryptophan and other aromatic compounds can undergo pi stacking interactions with each other

protein primary structure

order of amino acids, sequence, N-C synthesis, defined by peptide bonds

nonpolar sequences prefer to be on the inside of a protein or in the transmembrane region

protein secondary structure

alpha-helix is right-handed, 3.6 aa per turn, no prolines, favorable for transmembrane proteins, defined by hydrogen bonds between backbone components

beta-sheet is parallel or antiparallel, hydrogen bond

prolines and glycines are used for turns in protein structure

protein tertiary structure

interactions between residues in the chain

covalent:

disulfide bonds

non-covalent:

hydrophobic interactions- hydrophobic residues fold in the interior of the protein

polar interactions- van der Waals

ionic interactions- acid/base side groups

protein quarternary structure

interactions between residues between different polypeptides, allows connection of subunits to form protein

hydrophobic force

hydrophobic collapse- proteins fold to push hydrophilic sections to the exterior and hydrophobic sections to the interior

solvation shell- water molecules interact unfavorably with hydrophobic sections, so water molecules forced to lock their orientation and form shells around the protein

this low entropy and high free energy state is relieved by protein folding

protein unfolding

sigmoidal since its a cooperative process

determines thermodynamic stability

drugs

IC50 is concentration of drug that inhibits 50% of cells

Kd is dissociation constant between drug and target, with a lower value indicating higher affinity

drugs are metabolized by the body for excretion

vmax

all active sites on enzymes are occupied, vmax constant

kinetic measurement

depends on:

1. type of enzyme

2. concentration of enzyme

when there is high concentration of substrate. vmax is more important than Km

Km

concentration of substrate to reach 1/2 vmax

thermodynamic measurement

essentially the affinity of E for S, high affinity means low Km, depends on the properties of the binding site

always assume reversibility

if vmax is lowered than Km is lowered too

when there is low concentration of substrate, Km is more important than vmax

competitive inhibition

same vmax, increased km

inhibitor binds at active site

can always be out competed by additional substrate, so vmax doesn't change

noncompetitive inhibition

decreased vmax, same km

inhibitor binds at allosteric site

alters shape of active site, so vmax decreases

binds to enzyme and enzyme-substrate substrate with same affinity

uncompetitive inhibition

decreased vmax, decreased Km

inhibitors binds to ES complex, mixed inhibition type I

increased substrate increases inhibitor effectiveness

locks S in active site, so decreases vmax and km

mixed inhibition

decreased vmax, increased/decreased km

binds at allosteric site, better affinity than substrate

type I: prefers to bind ES complex, so decreased Km

type II: prefers to bind E alone, so increased Km

Michaelis-Menten equation

v = vmax[S]/(Km+[S])

vmax = k_cat*[E]

catalytic efficiency = k_cat/Km

Lineweaver-Burke plot

inverse of rxn speed is y

inverse of substrate conc. is x

1/Vmax is y-intercept

1/Km is x-intercept

useful for testing inhibitor's effect on vmax and Km, plot two lines with and without inhibitor

slope is Km/vmax

ternary complex mechanisms

both substrates occupy active site at same time:

1. ordered mechanism- one substrate must bind first

2. random order mechanism- doesn't matter which is first

specific activity

units of enzyme per total protein mg

use specific activity to calculate purity

use just the units of enzyme (specific activity times total protein) to calculate yield

cellular respiration

NAD (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) accept electrons by getting reduced, later get oxidized on delivery to ETC

glucose is oxidized to CO2, O2 is reduced to H2O

1. glycolysis occurs in the cytosol

2. PDC and TCA cycle occurs in mitocondrial matrix, except in prokaryotes where it occurs in cytosol

3. ETC and oxidative phosphoylation occurs on inner mitochondrial membrane, except in prokaryotes where it occurs on cell membrane

electron carriers

NADH -> NAD+ + H+ + 2e-

FADH2 -> FAD+ + H+ + 2e-

NADH and FADH2 carry 2 electrons

CoQ carries 1 or 2 electrons

cytochrome C carries 1 electron

glycolysis

all cells possess this pathway, occurs in cytoplasm

glucose is oxidized and split into two pyruvates, produced net 2 ATP, 2 NADH

3 key steps:

1. hexokinase- converts glucose to glucose-6-P, uses ATP

2. phosphofructokinase (PFK) - converts fructose-6-P to fructose-1,6-P2, committed step of glycolysis, allosterically inhibited by high ATP

3. pyruvate kinase- converts PEP to pyruvate, produces 2 ATP

"goodness gracious, father franklin did go buy phat pumpkins (to) prepare pies"

"GG, final fantasy did get boring playing people punching people"

steps in glycolysis that create/require energy

require ATP (2 ATP investment for 1 glucose):

1. hexokinase- glucose to glucose-6-P

2. PFK- fructose-6-P to fructose-1,6-P2

create ATP (4 ATP total, 2 ATP net for 1 glucose):

1. phosphoglycerate kinase- 1,3-bisphosphoglycerate to 3-phosphoglycerate

2. pyruvate kinase- PEP to pyruvate

create NADH (2 total for 1 glucose):

1. GAP DH- glyceraldehyde-3-P to 1,3-bisphosphoglycerate

fermentation

aerobic conditions- pyruvate enters Krebs cycle, NADH from glycolysis is oxidized in ETC

anaerobic conditions- 2 ATP produced, 2 NADH must go back to regenerate NAD+ to continue glycolysis

to regenerate NAD+, pyruvate reduced to ethanol (yeast) or lactate (muscle), toxic when building up

pyruvate dehydrogenase complex

oxidative decarboxylation, pyruvate oxidized to acetyl-CoA (loses a carbon)

uses up CoA, NAD+ reduced to NADH, releases CO2

TPP- prosthetic group which is a covalently bound cofactor that helps with decarboxylation, derived from thiamine (vitamin B)

thiamine deficiency would increase rate of anaerobic glycolysis

allosteric regulation- ATP and fatty acids inhibit, since acetyl-CoA goes to fatty acid synthesis and ATP synthesis

cofactors of pyruvate dehydrogenase complex

1. TPP- thiamine derived

2. lipoic acid

3. FAD+

4. NAD+ (converted to NADH, so technically not cofactor)

5. CoASH (attached to pyruvate to form acetyl-CoA)

TCA cycle

acetyl-CoA converted to citric acid, OAA from previous cycle also converted to citric acid

each turn produces 2 CO2, 3 NADH, 1 GTP, 1 FADH2

each glucose does two turns

aconitase- only enzyme name that doesn't match product

"can I keep selling sex for money, officer?"

regulation of TCA cycle

substrate availability- amino acids can be converted to alpha-ketoglutarate to speed up TCA cycle

substrates inhibit their own enzyme- citrate inhibits citrate synthase, succinyl-CoA inhibits aKG DH

allosteric regulation- ATP, NADH inhibit TCA cycle

steps in TCA cycle that create energy

create NADH (3 total for 1 turn, 6 total for 1 glucose):

1. pyruvate DH complex- pyruvate to acetyl-CoA (technically not part of TCA cycle)

2 isocitrate DH- isocitrate to alpha-ketoglutarate

3. aKG DH- alpha-ketoglutarate to succinyl-CoA

4. malate DH- malate to OAA

create GTP (1 total for 1 turn, 2 total for 1 glucose):

1. succinyl-CoA synthetase- succinyl-CoA to succinate

create FADH2 (1 total for 1 turn, 2 total for 1 glucose):

1. succinate DH- succinate to fumarate

oxidative phosphorylation

two steps:

1. ETC- empty the electron carriers

2. chemiosmosis- make ATP

3 complexes pump H+ to intermembrane space:

1. NADH dehydrogenase- converts NADH to NAD+, CoQ carries electrons to complex 3

2. converts FADH2 to FAD+, CoQ carries electrons to complex 3

3. cytC reductase- cytC carries electrons to complex 4

4. cytC oxidase- O2 accepts electrons, converts to H2O

ATP synthase- H+ flows allowed to flow from intermembrane space to matrix, converts ADP to ATP

NADH produces 3 (2.5) ATP, moves 10 H+

FADH2 produces 2 (1.5) ATP, moves 6 H+

energetics of glucose catabolism (ATP count)

1. glycolysis- 2 ATP, 2 NADH (5 - 2 to bring NADH into mitochondria = ~3 ATP)

2. PDC- 2 NADH (~5 ATP)

3. 2 GTP (2 ATP), 6 NADH (~15 ATP), 2 FADH2 (~3 ATP)

ideal total: 38 ATP per glucose (actual: 30 ATP)

prokaryotes ideal total: 38 ATP

anaerobic glycolysis: 2 ATP

gluconeogenesis

activated by low glucose, high ATP

requires 6 ATP, 2 NADH to convert pyruvate to glucose

slightly different from glycolysis because pyruvate kinase is irreversible, so instead pyruvate is converted to OAA, then to PEP

bypasses acetyl-CoA, which means fatty acids cannot be converted to glucose

first step by pyruvate carboxylase happens in mitochondria, then transported out to cytosol

formation of glucose, fructose-6-P, and PEP are irreversible steps that push equilibrium to favor gluconeogenesis

glycogen- stored in liver, converted to glucose

steps in gluconeogenesis that require energy

require ATP (6 total for 1 glucose):

pyruvate carboxylase- pyruvate to OAA

PEP carboxykinase- OAA to PEP

phosphoglycerate kinase- 3-phosphoglycerate to 1,3-bisphosphoglycerate

require NADH (2 total for 1 glucose):

GAPDH- 1,3-bisphosphoglycerate to glyceraldehyde-3-P

adding phosphate to glucose and fructose at the end does not require ATP

starting materials of gluconeogenesis

lactate, pyruvate, glycerol (enters through DHAP), amino acids (enter through pyruvate), any TCA cycle intermediates (enter through OAA)

glycogenolysis

glycogen is converted to glucose-6P

glycogen phosphorylase- just add phosphate group, no ATP required

regulation:

allosteric- ATP and glucose inhibit glycogenolysis

hormonal- epinephrine and glucagon activates glycogenolysis, insulin inhibits, all done through cAMP/pkA signalling

3 endpoints:

1. glycolysis- energy source for muscles

2. gluconeogenesis- glucose-6-phosphatase is only in live, converts to glucose and releases to blood

3. pentose phosphate pathway

regulation of cellular respiration

high ATP and citrate indicate Kreb's cycle activity, both inhibit PFK allosterically, activate fructose-1,6-P2ase (FBP)

substrate availability- glucose influx activate glycolysis, OAA influx activates gluconeogenesis

insulin- released with high glucose, activate PFK, promotes glycolysis, also recruits glucose transporters to plasma membrane, increases storage in glycogen and lipids

glucagon- released with low glucose, inhibit PFK, activate FBP, promotes gluconeogenesis, breaks down stored glycogen and lipids

pentose-phosphate pathway

starts with glucose-6-P getting converted by GAPDH

releases 2 NADPHs total

oxidative phase- glucose-6-P converted to ribulose-5-P, 2 NADPHs and CO2 produced

non-oxidative phase- ribulose-5-P converted to ribose-5-P and glycolysis intermediates (2 GAP, 2 fructose-6-P)

it takes 3 glucose-6-P to make it through both phases

3 goals:

1. NADPH for reducing power in fatty acid synthesis

2. NADPH for eliminating free radicals

3. ribose-5-P for producing nucleotides

fatty acid oxidation (beta oxidation)

saturated fatty acids- dehydrogenase to create double bond (produce FADH2), then produce NADH to create ketone, breaks off acetyl-CoA, repeat

unsaturated fatty acids- isomerase to move double bond, then produce NADH to create ketone, break off acetyl-CoA, repeat

in mitochondrial matrix

needs 2 ATP to initially activate fatty acid

need 1 FAD, 1 NAD+ for each 2 C removed

produces 1 FADH2, 1 NADH

fatty acid metabolism

energy stored as triglycerides, glycerol, fatty acids

lipase is enzyme that breaks down triglycerides

1. in cytosol, fatty acid activated by addition of S-CoA to carboxylic end

2. in matrix, fatty acid undergoes beta oxidation to acetyl-CoA

3. goes to TCA cycle

fatty acid ketogenesis

during starvation, glucose level fall and fatty acids are oxidized to supplement TCA cycle

in liver cells, remaining acetyl-CoA produced react together to form ketone bodies, enter brain or other organs to be reconverted to acetyl-CoA

2 acetyl-CoAs combined to form acetoacetate, which can split to beta-hydroxybutyrate and acetone

fatty acid synthesis

starts with acetyl-CoA and malonyl-CoA (from acetyl-CoA, using bicarbonate), activated to acetyl-ACP and malonyl-ACP

acetyl-ACP to acetyl-FAS (with fatty acid synthase attached)

fatty acid synthase helps combine malonyl-ACP with acetyl (release CO2), NADPH to remove ketone, then NADPH to remove double bond

in cytosol

need 2 NADPH for each 2 carbons added

protein catabolism

protein broken down to amino acids by proteases

3 endpoints:

1. can be used to construct other proteins

2. amino end can be used for nucleotides or urea (excretion)

3. remaining carbon skeleton can be converted to acetyl-CoA or glucose

metabolic rate

how quickly an organism uses up stored energy reserves (protein, lipids, sugars)

metabolic states

absorptive state- glucose storage as glycogen in liver, fatty acid storage as triglycerides in adipose tissue, brain and muscle still using up glucose

post-absorptive state- glycogen broken down to glucose in liver, triglycerides broken down to acetyl-CoA to power TCA cycle and ketogenesis (which can enter brain)