Unit 1 Flashcards
Gibbs Free Energy (🔺G)
A negative 🔺G means a reactions is energetically favorable (exergonic; i.e., gives off energy)
Enthalpy (🔺H)
A negative 🔺H means heat is released (exothermic)
Entropy (S)
Randomness; randomness is energetically favorable, order is NOT energetically favorable
Equilibrium Constant (Keq)
Measurement of how far a reaction proceeds in a net direction until equilibrium is reached; a large Keq means that at equilibrium, almost all reactant will have been converted to product
⬆️Keq = ⬇️(more negative) 🔺G
Hydrogen Bonding in Ice
In ice, water forms 4 H-bonds/molecule. Heat collapses the crystalline structure of ice, establishing a transient effect of breaking/forming bonds; ice represents water in its most expanded state
Directional preference in Hydrogen bonding
Linear preference because nonbonded electrons are in alignment; greater distance weakens bond strength
Hydrophobic Effect
Dispersed lipids surrounded by ordered water (entropically unfavorable), lipids cluster and release water (entropically favorable); spontaneous clustering of non-polar groups maximizes the entropy of water
Calculating hydrogen and hydroxide ion concentration
Kw=[H+][OH-]=1.0x10^-14
pH
Dictates acidity/basicity
pH=-log[H+]
pKa
Measure of acid strength
Ka=[H+][A-]/[HA]
pKa=-log[Ka]
Henderson Hasselbach Equation
pH=pKa+log([A-]/[HA])
Buffer Region
Enough acid and base creates buffer region where pH remains relatively unchanged; [HA]=[A-]
Commonality in all amino acids
Alpha carbon with COO- group, NH3+ group, H group, and R group
Zwitterionic Form
State of amino acid where net charge=0
Isoelectric Point
Point where Zwitterion dominates (i.e., where net charge=0)
Peptide Bond Formation/Breakage Reaction
Peptide bond formation is a condensation reaction; AA + AA ➡️ Peptide + H2O
Peptide bond breakage is a hydrolysis reaction; Peptide + H2O ➡️ AA + AA
Deriving Isoelectric Point
Write out peptide in a table with ionizable end groups, choose pH range and depict charge at each pH (pKa>pH means proton won, pKa
UV Light Protein Purification
Tryptophan (strong signal), and Tyrosine (weak signal) absorb UV light
Ion Exchange Chromatography
Protein mixture is added to column containing cation exchangers. Proteins move through column at rates determined by their net charge at the pH being used. With cation exchangers, proteins with large net negative charge move faster and elute earlier; elution is achieved by changing salt conditions
Size Exclusion Chromatography
A porous column acts as a molecular sieve and protein molecules separate by size. Larger molecules pass first
Affinity Chromatography
Solution of ligand is added to column. Protein mixture is added to column. Protein binds to ligand (ATP) and is extracted. Protein that doesn’t bind is unwanted and removed. Elution is achieved with a high concentration of free ligand.
Specific Activity
Measures protein specificity (purity); calculated from Activity(units)/Total protein(mg)
Electrophoresis (SDS-Page)
Negative sulfate group of SDS is exposed, and protein is coated in negative charge. Negative charge causes protein to migrate toward a positive charge. Large proteins move slowly through gel, small proteins move quickly
Isoelectric Focusing
A protein sample may be applied to one end of a gel strip. After staining, proteins are shown to be distributed along pH gradient according to their pI values; low pI, lots of acidic groups (lots of negative charge), means protein migrates further toward positive terminal
Mass Spectrometry
Get molecules to “fly” in the gas phase by electrospray ionization. Separate ions by mass in a vacuum. Lighter ones go farther
Tandem Mass Spectrometry
Can be used to sequence a protein by identifying fragments of unique mass. Once fragments are determined, they can be back-converted into the corresponding DNA sequence. The entire protein sequence can be deduced from overlapping fragments
Primary Structure of Protein
Amino acid residues; linear structure, sequential order
Secondary Structure of Protein
Alpha helix and beta sheet configuration
Tertiary Structure of Protein
Polypeptide chain; series of secondary structures joined
Quaternary Structure of Protein
Assembled subunits; more than one polypeptide chain joined together
Why is protein sequencing important?
- can be used to identify a protein of interest
- identify mutations involved in disease
- understand shape and function (via homology to other similar proteins)
shape of peptide bond
planar due to the partial double-bond of the carbonyl carbon-amide nitrogen bond (carbonyl oxygen has a partial negative charge and the amide nitrogen has a partial positive charge, setting up a small electric dipole)
How do proteins fold and take shape?
rotation of two bond angles, phi and psi, in the peptide backbone; peptide bond is planar and the bonds on either side can rotate
Ramachadran plot
displays allowed regions of protein folding space
alpha helix
secondary structural motif
Properties:
- Right handed
- 3.6 amino acids/turn
- H-bond between C=O(n)…H-N(n+4)
determining number of hydrogen bonds in alpha helices
n-4, where n is the number of amino acids
beta sheets
secondary structural motif; made up of beta strands and can be either parallel or antiparallel
- hydrogen bonds are formed between strands
- side chains are on alternate sides of the sheet to form a pleated sheet
- sheets are not flat; they have a characteristic twist
- strands contain relatively few amino acid residues (3-10)
antiparallel beta sheets
R groups project outward in alternating directions, but strand direction alternates; linear hydrogen bonds are formed, making antiparallel beta sheets stronger
parallel beta sheets
R groups project outward in alternating directions, but strand direction is consistent; hydrogen bonds are formed at an angle, making parallel beta sheets weaker
2 general classes of protein structure
fibrous and globular
fibrous proteins
highly extended and exhibit repeating helical or beta sheet structure (e.g., keratin and collagen)
keratin
fibrous protein in hair, skin, feathers, and nails
Properties:
- extended alpha helices, cross linked by disulfide bonds
- composed of many hydrophobic residues
- high tensile strength
collagen
fibrous protein in bone, cartilage, and connective tissue
Properties:
- triple helix of a polymer with repeating motif (Gly, Pro, HyPro)
- most abundant protein in humans
- high tensile strength
hydroxyproline
post-translational modification that is required for collagen to form a stable coiled-coil structure
protein stability
the difference in free energy between the folded and unfolded state; the major source of protein stability is the hydrophobic effect, as the sequestering of hydrophobic side chains into the interior of the protein in the folded state releases ordered water (entropy of water increases as water is released)
structure of water-soluble folded proteins
hydrophobic side chains are oriented towards the interior of the protein, while polar and charged side chains are oriented towards the outer surface, forming a hydrophobic core
protein size limit
most have molecular weight less than 100,000 Daltons (1,000 amino acids)
Reasons:
1) It is more efficient to build large structures from lots of smaller ones (less energy required)
2) The error rate of protein synthesis is 1 mistake per 10,000 amino acids
myoglobin
oxygen storage protein found in muscles and composed of 153 amino acids (8 alpha helices) that surround heme
heme
consists of porphyrin coordinated to an iron atom; porphyrin ring is flat (aromatic) and provides 4 nitrogen ligands to the iron, which helps stabilize the Fe2+ vs Fe3+ state
How is Fe2+ stabilized in myoglobin?
the protein fold; Fe3+ does not bind O2, and oxidation of Fe2+ to Fe3+ is prevented by sequestering the heme inside the protein
oxygen binding site in myoglobin
oxygen binds at an angle of 120 degrees to Fe; oxygen hydrogen bonds to the distal histidine (histidine farthest from Fe)
myoglobin binding specificity
the protein fold influences ligand binding, as the protein’s distal histidine H-bonds to O2 and improves binding specificity; because carbon monoxide binds to free heme 20,000x stronger than O2, this fold is extremely significant
P50
the partial pressure of oxygen for 50% saturation; same thing as Kd
oxygen binding curve for myoglobin
fraction of myoglobin bound to oxygen vs partial pressure of oxygen; binding curve is hyperbolic
Mb + O2 –> MbO2
fraction of protein bound formula
theta=[L]/[L]+[1/Ka]
1/Ka=Kd, where Kd is the dissociation constant
When [L] is equal to Kd, half of the binding sites are occupied
Kd
dissociation constant, depicts oxygen binding affinity; low Kd means tighter binding
hemoglobin
oxygen transporter in blood; formed from two different subunits, alpha and beta, to form a tetramer (four subunits)
hemoglobin vs myoglobin
hemoglobin is the oxygen transporter in blood, whereas myoglobin is present in muscle tissue; hemoglobin is a tetramer (comprised of four subunits) whereas myoglobin is a monomer
hemoglobin T state
predominates in tissues, has lower fractional saturation (64% O2 bound); primarily present when releasing O2
hemoglobin R state
predominates in the lungs, has higher fractional saturation (96% O2 bound); primarily present when binding O2 in the lungs
Helix F and conversion between T and R states
oxygen binding moves the proximal histidine which pulls on helix F which changes the conformation of the interface from T state to R state
hemoglobin oxygen binding curve
sigmoidal as the result of cooperativity between the four subunits of hemoglobin
cooperativity
binding of the first molecule allows subsequent molecules to bind more tightly; provides a route towards regulating the affinity of hemoglobin for oxygen through interactions with other ligands (the case for hemoglobin)
hemoglobin H+ transportation
H+ is bound to several side chains whose pka’s are altered by the transition from R to T
hemoglobin CO2 transportation
CO2 is transported by carbamylation of the amino-terminal amino acid
Bohr Effect
idea that the binding affinity of hemoglobin for oxygen decreases at lower pH; low pH stabilizes the T state, favoring the uptake of protons and release of O2 in the tissue
2,3-BPG
binds in the cavity between the subunits in the T state (in the R state this cavity is blocked by His) and regulates the binding affinity of hemoglobin for oxygen; without BPG the curve is hyperbolic, there is no cooperativity (R state)
allosteric protein
ligand binding induces a conformational change (e.g., hemoglobin)
BPG at high altitude
at high altitude, there is more BPG and thus more hemoglobin in the T state and more oxygen delivered to the tissues
enzymes
catalyze reactions by lowering the activation energy of the transition state
enzymatic general acid-base catalysis
often the enzyme provides additional functional groups that aid in catalysis once the substrate is bound
enzymatic covalent catalysis
characterized by the formation of a covalent bond between the enzyme and substrate at some point during catalysis; covalent bond must be broken later in catalytic cycle in order to release product and regenerate free enzyme
enzymatic metal ion catalysis
metal ions have a positive charge that stabilizes negatively charged transition states
enzyme equilibrium constant
E+S <-> ES <-> EP <-> E+P
Keq = [P]/[S]
Michaelis Constant (Km)
equal to the concentration of substrate [S] at 1/2Vmax; lower Km means greater efficiency (i.e., more tightly binded to substrate, less substrate required to yield product)
enzyme kinetics
visualized via Initial Velocity V0 vs [S] graphs to provide fundamental insight into the chemical mechanism of enzyme catalysis
Michaelis Menton equation
simple algebraic relationship between the substrate concentrations and initial rate (V0); it is the rate equation for an enzymatic reaction
V0 = Vmax[S]/(Km+[S])
Km = (k2 + k-1)/k1
kcat
first order rate constant (time-1) that is often called the turnover number; generic rate limiting step
catalytic efficiency
can be quantitated by kcat/Km; larger kcat, smaller Km ideal for efficiency
for very efficient enzymes, diffusion becomes the rate limiting step
chymotrypsin mechanism in 7 steps
1) Substrate binds in hydrophobic pocket, forming enzyme-substrate complex
2) Histidine acts as a base to activate serine by deprotonating hydroxyl group. Serine performs covalent catalysis by having its alkoxide ion attack a carbonyl carbon of the substrate, forming a covalent acyl bond between enzyme and substrate. Tetrahedral TS involving oxyanion is stabilized by the oxyanion hole.
3) Histidine acts as a general acid to protonate an amine nitrogen. The peptide bond is broken, causing the TS to collapse and causing the release of the first product.
4) Water enters the active site and is converted into a hydroxide ion by a histidine acting as a general base. The hydroxide ion attacks the acyl bond between substrate and enzyme.
5) A tetrahedral transition state involving an oxyanion is stabilized by the oxyanion hole.
6) Collapse of TS intermediate forms the second product.
7) Histidine acts as a general acid to protonate the serine oxygen group, breaking the acyl bond between enzyme and substrate. The second product dissociates.
serine in chymotrypsin mechanism
covalent catalysis (acylation)
histidine in chymotrypsin mechanism
general acid-base catalysis; acts as a base to activate serine by deprotonating hydroxyl group and acts as an acid by transferring proton to leaving group; also deprotonates water and transfers proton back to serine oxygen
aspartate in chymotrypsin mechanism
hydrogen bonds to His to stabilize positive charge on His
hydrophobic pocket in chymotrypsin mechanism
substrate binding and specificity
oxyanion hole in chymotrypsin mechanism
created by hydrogen bonds from the N-H group of serine and glycine; lowers activation energy by stabilizing oxyanion in transition state
ribozyme
enzyme that is a ribosome; active site made entirely of RNA
regulatory enzymes
enzymes that exhibit increase or decreased activity in response to certain signals
3 types:
1) Allosteric enzymes
2) Covalently modified enzymes
3) Zymogens
allosteric enzymes
shape-changing; bind regulatory compounds (allosteric modulators) non-covalently (reversibly); have quaternary structure with both catalytic and regulatory subunits
have non-Michaelis-Menten behavior (sigmoidal instead of hyperbolic kinetics)
covalently modified enzymes
regulatory compounds are covalently attached in a reversible manner; involves post-translational modification (commonly phosphorylation)
zymogens
enzymes made as inactive precursors that must be cleaved to become active
reversible enzyme inhibitors
bind in or close to the active site; competitive, uncompetitive, and mixed
irreversible enzyme inhibitors
covalently attach to enzyme, also called suicide substrates
Lineweaver-Burke Plots
inverse of Michaelis-Menten equation; linear plot instead of hyperbolic
1/V0 = Km/Vmax[S] + 1/Vmax
competitive inhibition
whichever arrives first, substrate or inhibitor, binds and takes effect; Vmax remains unchanged, while Km increases upon inhibition
uncompetitive inhibition
substrate binds at active site, then inhibitor binds adjacent to active site, inactivating the ES complex and forming ESI complex; Km and Vmax decrease equally
mixed inhibition
elements of both competitive and uncompetitive inhibition; Km increases while Vmax decreases
biological lipids
a diverse class of organic molecules that share the common feature of insolubility in water; function as forms of energy stores, biological membranes, or hormones and messengers
packing of saturated vs unsaturated fatty acids
saturated fatty acids are linear and thus packed more densely than unsaturated fatty acids; more energy is required to disrupt this
melting points of unsaturated vs saturated fatty acids
the melting point of unsaturated fatty acids is significantly lower than that of saturated fatty acids because unsaturated fatty acids are packed together less densely and can thereby be disrupted with less energy; hydrophobic effect increases with tail length, giving longer saturated fatty acids a higher melting point as well
triacylglycerols
fatty acids attached to glycerol through ester linkages; principal component of fat cells, used for long term energy storage
What makes triacylglycerols an efficient source of energy?
1) They are highly reduced
2) Provide >2x the energy as carbohydrates
3) They are dehydrated and hence take up less space
Sole disadvantage is that they are metabolized more slowly than glycogen, starch, or other carbohydrates.
three major classes of lipids found in membranes
1) Glycerophospholipids
2) Sphingolipids
3) Sterols
glycerophospholipids
have glycerol backbone, two fatty acids, and PO4/alcohol
sphingolipids
nearly identical to glycerophospholipids but have different backbone that results in one less fatty acid; important immunogenic determinatant in blood, as the sugar that sticks out of membrane dictates blood type
prostaglandins
complex group of molecules that influence a wide range of biological functions, including the inflammatory response and pain and fever; derivatives of glycerophospholipids containing arachidonic acid
prostaglandin synthesis
arachidonic acid is cut from glycerophospholipids and converted via enzymatic action into prostaglandins
sterols
type of membrane structural lipid; major sterol is cholesterol
hormones derived from cholesterol
testosterone, estradiol, cortisol, aldosterone, prednisolone, prednisone
vitamin derived from cholesterol
Vitamin D
function of vitamin D
precursor to a hormone that regulates calcium uptake in bone
liposomes
spherical particles formed when hydrophobic heads form an outer layer as well as an aqueous central cavity
How are lipids distributed in membranes?
asymmetrically; equilibrium not possible because of membrane fluidity
membrane fluidity
controlled by regulation of lipid content in the bilayer; as temp increases, membrane contains more saturated fatty acids to prevent TOO MUCH fluidity (i.e., membrane made stronger and hydrophobic effect is kept in check)
removal of integral membrane proteins
requires detergents
two classes of membrane proteins
1) Peripheral membrane proteins
2) Integral membrane proteins
peripheral membrane proteins
associated with the membrane, dissociated by gentle means
integral membrane proteins
tightly associated with the membrane, require detergent for removal, inserted in one orientation, never flip-flop spontaneously; transmembrane regions are hydrophobic, anchoring protein into the bilayer
hydropathy plots
reveal hydrophobic/hydrophillic regions of transmembrane alpha helices; can be used to predict membrane proteins
beta barrel membrane proteins
built almost entirely from beta strands, have hydrophobic residues that face lipid bilayer and hydrophillic residues that line the pore and upper and lower outer surfaces; allow selective diffusion of ions and small molecules
passive transport
diffusion down concentration gradient without an energy requirement; can be simple diffusion or facilitated diffusion
active transport
diffusion against a concentration gradient with an energy requirement; can be primary or secondary
simple diffusion
diffusion directly across a membrane without use of protein (e.g., O2 diffusion)
facilitated diffusion
requires participation of protein carrier; can be facilitated by channels or passive transporters
primary active transport
requires energy (e.g., ion pumps)
secondary active transport
uses gradient established by primary active transport to co-transport another solute
Na+/K+ pump
example of primary active transport; pumps K+ into the cell and Na+ out of the cell, both against their concentration gradients
