Structure and Function of Proteins

Structure and function of proteins and their constituent amino acids Amino Acids (BC,OC) • • Description o Absolute configuration at the α position ▪ The alpha carbon IN EVERY amino acid is a chiral center EXCEPT in glycine (it is achiral, since the R group is an H) ▪ EVERY AA has S configuration EXCEPT FOR cysteine (R configuration) o Amino acids as dipolar ions ▪ At low pH, amino acid = cationic ▪ At high pH, amino acid = anionic ▪ At pH = pI, amino acid = zwitterionic (neutral) o Classifications ▪ Acidic or Basic • ACIDIC: Aspartic Acid (Asp, D) ; Glutamic Acid (Glu, E) • BASIC: Lysine (Lys, K) ; Arginine (Arg, K) ; Histidine (His, H) ▪ Hydrophobic or Hydrophilic • HYDROPHILIC: If the R group contains acids, bases, amines or alcohols o Arginine (Arg, R), Lysine (Lys, K), Aspartic Acid (Asp, D), Glutamic Acid (Glu, E), Glutamine (Gln, Q), Asparagine (Asn, N), Histidine (His, H), Serine (Ser, S), Threonine (Thr, T), Tyrosine (Tyr, Y), Cysteine (Cys, C), Tryptophan (Trp, W) • HYDROPHOBIC: If the R group DOES NOT contain what is listed above ^^ o Alanine (Ala, A), Isoleucine (Ile, I), Leucine (Leu, L), Methionine (Met, M), Phenylalanine (Phe, F), Valine (Val, V), Proline (Pro, P), Glycine (Gly, G) Reactions o Sulfur linkage for cysteine and cystine ▪ Cysteine = amino acid with the thiol R group ▪ Cystine = 2 cysteines that have formed a disulfide bond o Peptide linkage: polypeptides and proteins ▪ Peptide bonds link amino acid chains together ▪ Peptide bonds are formed by the nucleophilic addition-elimination (condensation, dehydration rxn) reaction between the carboxyl group of one amino acid and the amino group of another amino acid ▪ The nucleophilic amino group attacking an electrophilic carbonyl ▪ The bond when formed has a lot of resonance delocalization (partial double bond character all over the place!) • Makes the bond very rigid/planar • However, this is still free rotation around the ALPHA CARBON o Hydrolysis ▪ The process of breaking the peptide bond • Done by either acid/base hydrolysis (nonspecific) or with the help of proteolytic enzymes (specific) Protein Structure (BIO, BC, OC) • Structure o 1o structure of proteins ▪ Linear sequence of amino acids ▪ Determined by the peptide bond linking each amino acid ▪ Covalent (Peptide) bonds o 2o structure of proteins ▪ Local structure, stabilized by hydrogen bonding • α-helices – hydrogen bonds run up and down, stabilizing the structure • β-pleated sheets – stabilized by hydrogen bonds connecting the sheets o Antiparallel vs. Parallel configurations ▪ The way the linear sequence folds on itself ▪ Determined by the backbone interactions (primarily hydrogen bonds) ▪ Hydrogen bonds between backbone atoms o 3o structure of proteins ▪ 3-D structure stabilized by hydrophobic interactions, acid-base interactions (salt bridges), hydrogen bonding, and disulfide bonds ▪ Depends on distant group interaction • Stabilized by hydrogen bonds, van der Waals, hydrophobic packing, disulfide bridge formation ▪ Disulfide bond formation happens on the exterior of the cell (covalent bond of two cysteines) • Extracellular space prefers the formation of disulfide bonds (the oxidizing environment) ▪ Hydrophobic interactions and polar interactions between side chains o 4o structure of proteins ▪ Interactions between subunits (multiple polypeptides) ▪ Hydrophobic interactions and ionic bonds between side chains (i.e. cysteine side chains making disulfide bonds) • Conformational stability o Denaturing and Folding ▪ Primary Structure = determined by peptide bonds ▪ Secondary Structure = determined by backbone interactions (hydrogen bonds) ▪ Tertiary Structure = determined by distant interactions between groups (van der Waals, hydrophobic packing, disulfide, hydrogen bonding) ▪ Quaternary Structure = determined by same bonds from tertiary structure ▪ Protein is ONLY FUNCTIONAL when in the proper conformation • A force that helps stabilize the protein is the solvation shell o Solvation shell = layer of solvent surrounding the protein (can be the water solvent interaction with polar AAs, etc.) ▪ Denaturation = when a protein loses active conformation and becomes inactive • Occurs by changing pH, temp, chemicals or even enzymes ▪ If you denature by heating, you destroy all the structures of the protein except the primary structure (primary structure is conserved) o Hydrophobic Interactions/Solvation Layer (entropy) (BC) ▪ The hydrophobic regions of the protein aggregate, which releases the water from cages → This increases the entropy of water, which is the major thermodynamically favorable component of protein folding • Separation techniques o Isoelectric point ▪ pI is determined by averaging the pKa values that refer to the protonation and deprotonation of the zwitterion ▪ Isoelectric focusing = gel electrophoresis method that separates proteins on basis of their relative contents of acidic and basic residues (gel with pH gradient is used) o Electrophoresis ▪ Positively charged anode at bottom, negatively charged cathode at top ▪ Larger molecules will have harder time moving, thus separation created by size with the smallest molecules towards the bottom ▪ Native Page = retains structure of protein ; SDS-Page = break into subunits Non-Enzymatic Protein Function (BIO, BC) • Binding (BC) o Bind various biomolecules – bind specifically and tightly o Receptors/Ion channels in the membrane: ▪ Receptors bind or receive signaling molecules (ligand) which makes a chemical response (i.e. insulin receptor) ▪ Ion channels can allow ions to enter/exit the cell • Immune System o Antibodies = protein components of the adaptive immune system whose main function is to find foreign antigens and target them for destruction o Antigen = the ligand for antibodies ▪ Antigens can be thought of as little red flags for the immune system letting us know, “Hey, that’s not supposed to be there!” • Motors o Transport: e.g. Hemoglobin (at high concentration of ligand, have high affinity, at low concentration of ligand, have low affinity) o Myosin/Kinesin/Dynein ▪ Myosin = responsible for forces exerted by contracting muscles ▪ Kinesin/Dynein = motor proteins responsible for intracellular transport • Dynein = plays a role in the motility of cilia Enzyme Structure and Function (BIO, BC) • Function of enzymes in catalyzing biological reactions o Enzymes function to lower the activation energy of reactions (do not get used up!) o Structure determines function → change in structure = change in function • Enzyme classification by reaction type o 6 Types of Enzymes: ▪ Transferase • Move a functional group from one molecule to another • A + BX → AX + B ▪ Ligase • Join two large biomolecules, often of the same type • A + B → AB ▪ Oxidoreductase • Catalyze oxidation-reduction reactions that involve the transfer of electrons • Oxidase = oxidizing or taking away electrons from a molecule • Reductase = reducing or giving electrons to a molecule • A + B: → A: + B ▪ Isomerase • • • • • • • Interconversion of isomers, including both constitutional and stereoisomers • A→ B ▪ Hydrolase • Cleavage with the addition of water • A + H2O → B + C ▪ Lyase • Cleave without the addition of water and without the transfer of electrons (reverse reaction, synthesis, is usually more biologically important) • A → B + C (does not use water, or oxidation/reduction) • Lyases generate either a double bond or a ring structure Reduction of activation energy o Acid/Base catalysis = enzymes use acidic/basic properties to make rxns go faster by proton transfer o Covalent catalysis = enzymes covalently bind to help with electron transfer o Electrostatic catalysis = charged molecules or metal ions used to stabilize big positive or negative charges o Proximity/Orientation effects = enzymes make collisions between reacting molecules happen more often o Transition state = highest energy point from path A to B (in A→ B) ▪ Where you also find most instability (high energy = more unstable) o Enzymes lower the activation energy of the reaction (making it easier for the reactants to transition to form products) Substrates and Enzyme Specificity o Enzyme-substrate specificity derives from structural interactions o Enzymes can be specific enough to determine between stereoisomers Active Site Model o Location on the enzyme where it reacts with its substrate o Shape/characteristics (functional groups) of an active site are responsible for the specificity of the enzyme Induced-fit Model o Initial Binding = when the substrate first binds to the enzyme (not perfect) ▪ Forces holding the two together are strong, but not at the maximum strength yet o Enzyme and substrate thus mold their shape to bind together super tightly ▪ Called the induced fit because both the enzyme and substrate have changed their shape a little so they bind together really tightly (catalyzing reaction at full force) o Binding between reactant and enzyme STRONGEST at the transition state Mechanism of catalysis o Cofactors ▪ Directly involved in the enzyme’s catalytic mechanism (might be stabilizing the substrates, or helping the reaction to convert substrates from one form to another) (e.g. Mg2+) o Coenzymes ▪ Organic carrier molecules (i.e. NADH, CoA) o Water-soluble vitamins ▪ Need to obtain from the diet ▪ Vitamins → organic cofactors and coenzymes ▪ e.g. Vitamin B3 is precursor for NAD ▪ e.g. Vitamin B5 is precursor for CoA Effects of local conditions on enzyme activity o Enzymes work best in specific environments o Effects of pH changes: e.g. DNA → Negatively charged → DNA Polymerase binds Mg2+ cofactor to stabilize negative charge on DNA • In normal conditions, DNA Pol holds onto Mg ion through electrostatic interaction between magnesium and one of its aspartate residues, which would be deprotonated and thus negatively charged at neutral pH values • If you took DNA Pol and put it in environment with reduced pH, the aspartate residue would become protonated since pH has dropped so much, and protonated form has no negative charge, so can’t bind Mg ion cofactor • DNA Pol cannot do job properly in low pH environment o Effects of temperature changes: ▪ Proteins fold from primary → secondary → tertiary → quaternary structures to function properly ▪ Significant changes in temp cause protein to lose its functionality (loses its shape) • e.g. when we get sick and our body temperature goes up, our digestive enzymes cannot work properly and consequently we cannot eat food as well ▪ Control of Enzyme Activity (BIO, BC) • Kinetics o General (catalysis) ▪ Enzymes lower the activation energy of a reaction, or the ΔG of the transition state (NOT OF THE RXN!) ▪ E + S → ES → E + P ▪ At really high [S] the enzymes will be saturated • Even if you increase concentration of [S] from this point, there will still be a Vmax o Michaelis-Menten ▪ Vmax is defined for a specific enzyme concentration (adding more enzyme will increase the Vmax) ▪ Michaelis-Menten equation calculates the rate of reaction using Vmax, the substrate concentration [S], and the Michaelis constant Km. Km = the [S] required to reach 1/2Vmax. ▪ Km does not fluctuate with changes in [enzyme] and is indicative of enzyme-substrate affinity ▪ Enzymes with high enzyme-substrate affinity will reach 1/2Vmax at a lower substrate concentration (Lower Km) ▪ Lower enzyme-substrate affinities will result in needing a higher substrate concentration to reach 1/2Vmax (Higher Km) 𝑣𝑚𝑎𝑥 [𝑆] ▪ V= 𝐾𝑚+[𝑆] • As substrate concentration increases, the reaction rate also increases until a maximum value is reached • At ½ Vmax, [S] = Km ▪ kcat = Vmax / [E]T • = Enzyme’s “Turnover Number” • How many substrates can this enzyme turn into product in one second at its maximum speed ▪ Catalytic Efficiency = kcat / Km o Cooperativity ▪ Some proteins can bind more than 1 substrate ▪ Cooperativity = substrate binding changes substrate affinity ▪ Positive Cooperative Binding = Substrate binding increases affinity for subsequent substrate ▪ Negative Cooperative Binding = Substrate binding decreases affinity for subsequent substrate ▪ Non-Cooperative Binding = Substrate binding does not affect affinity for subsequent substrate ▪ TOW RIGH (Hemoglobin affinity for O2) • T state = Low affinity • R state = High affinity o Feedback Regulation ▪ When product of reaction binds allosteric site of the enzyme, affecting the catalytic activity • Can be positive = increases enzyme-substrate affinity • Can be inhibitory = reducing activity at the active site or inactivating it completely o Inhibition – Types ▪ Competitive • E (inhibitor binds to E here to make EI) + S → ES → E + P • Blocks the enzyme and makes it unable to react with substrate to form product • Inhibitor competes with substrate for space on the enzyme • Binds: Active Site • Impact on Km: Increases • Impact on Vmax: No Change ▪ Uncompetitive • E + S → ES (inhibitor binds to the ES here to make ESI) → E + P • Molecule that binds only to the enzyme-substrate complex, rendering it catalytically inactive • Binds: Allosteric Site • Impact on Km: Decreases • Impact on Vmax: Decreases ▪ Non-competitive • Prevents the enzyme from turning substrate into product • Binds to an allosteric site on the enzyme, causing a conformational change that decreases catalytic activity at the active site regardless of whether a substrate is already bound • Binds: Allosteric Site • Impact on Km: No Change • Impact on Vmax: Decreases ▪ Mixed • Molecule that binds to an allosteric site on the enzyme, causing a conformational change that decreases catalytic activity at the active site • Generally, have preference towards binding either the enzyme-substrate complex, or binding the enzyme alone • Binds: Allosteric Site • Impact on Km: Increase (if prefer enzyme w/o substrate) or Decrease (if prefer enzyme with substrate bound) • Impact on Vmax: Decreases o Regulatory Enzymes ▪ Allosteric Enzymes • Allosteric site present, molecule binds it, can either upregulate or downregulate the enzyme function ▪ Covalently-modified enzymes • Not all enzymes are proteins (i.e. Inorganic metals, small organic molecules like Flavin) • Small Posttranslational Modifications: o Translation → synthesis of AA polymer o “Post-translation” → after initial synthesis o “Small” → adding or removing small functional groups • Methylation o Modification of a protein that involves addition of methyl group (CH3) • Acetylation o Modification of a protein that involves addition of an acetyl group • Glycosylation o Addition of a sugar to a protein • I.e. Acetylation of lysine residue on a protein o Electron withdrawing impact of the acetyl group will prevent nitrogen from carrying positive charge and modify the behavior of the amino acid • Suicide Inhibition o Suicide inhibitors covalently bind the enzyme and prevent it from catalyzing reactions o Rarely unbind – why it’s called suicide (enzyme won’t work anymore) ▪ Zymogens • Inactive form of an enzyme that requires covalent modification to become active • I.e. Digestive enzymes of the pancreas o Pancreas releases trypsinogen (a zymogen) o Once in the intestine, it is covalently modified by an enzyme called enterokinase to the active form Trypsin o This makes sure trypsin does not break down proteins that we need in the pancreas