Biochem 1

ionizable side chains

5 amino acids that are bases and acids tyrosine cystine able to form ionic bonds as well as to donate or accept protons (called acid- base catalysis) to facilitate reactions

buffers

A buffer is a mixture of an undissociated acid and its conjugate base. Ex. Acetic acid and acetate ion Consequence: It causes a solution to resist changes in pH when either H+ or OH is added, only in small area combines with H+ or OH- and converts them to a non-ionized form, involves 2 reversible equilibria, one involving water

Formation of a Peptide Bond

A condensation reaction where water is eliminated. water is product, covalent bond, kinetically stable Carried out by catalytic RNA in ribosome to ensure bond stays, since reactants are favored means energy required Reaction is not favorable (requires an input of energy)

Hemoglobin Function

Hb must bind oxygen in lungs and release it in capillaries When the oxygen binds to Fe in heme of one Hb subunit, Fe is drawn into the plane of the porphyrin ring, makes radius of fe smaller This disrupts key noncovalent interactions in that subunit which causes a change in conformation 2 Additional coordination sites for Fe2+ : • 5 th site—proximal Histidine • 6 th site---binds O2 distal histidine prevents carbon monoxide binding to iron to create toxins, binds to o2 bond on histidine by h bonding, reduces probabilit co will outcompete

structure of heme

Heme = Protoporyphyrin ring + Fe2+ • Protoporphyrin ring [structure (-) Fe], ring allows iron to be fe2+ so o2 can react with it Hydrophobic, planar ring stucture so it can bind to hemoglobin and myoglobin to create hydrophobic pocket series of nonpolar amino acids 4 Nitrogens coordinate the Fe2+ and prevents Fe2+ to Fe3+ through their electron donating capacity (Fe2+ binds oxygen reversibly, Fe3+ can not) Heme is located within hydrophobic pocket of the structureprevents full transfer of electrons to give irreversible oxidation; key to understanding binding / release of O2. Free heme does not bind O2 reversibly

Amyloid

Highly ordered β-strands perpendicular to fiber axis (transition from normal α-helix to abnormal βsheet conformation); extensive H bonding, resistant to degradation very stable Importance of side chain interactions in aggregate formation Self assemble into fibers Resistant to degradation "Steric zipper" amyloid forms due to intrinsically disordered proteins becoming disordered

polar vs nonpolar

Equal sharing, uniform charge distribution: molecule is nonpolar Unequal sharing, asymmetrical distribution: molecule is polar because the bond involves an electronegative atom, hydrophillic

ion product constant for water

Kw=10^-14 [H+ ] increases, then [OH- ] decreases Kw = [H+] 2 = 1 x 10-7 water is neutral, pH=7 pH is defined as -log [H+], pH has 10x difference in h+

daily Metabolism produces changes in [H+ ]

Metabolism results in 13-22 moles of acid produced/day. If dissolved in water, pH would be < 1 Weak organic acids (HA) undergo partial ionization in water (solvent): HA + H2O------> H3O+ + A-

Thalassemias

are caused by a loss or substantial reduction of a single hemoglobin chain. - In α-thalassemia, the α chain is not produced in sufficient quantity. Tetramers of the β chain form (HbH) and bind oxygen with high affinity but no cooperativity. - In β-thalassemia, the β chain is not produced in sufficient quantity. The α chains aggregate and precipitate, leading to loss of red blood cells and anem

What is the relationship between ka and pH?

henderson hasselback eq relates weak acids and weak bases Ka is large, indicates strong acid Since pKa = -log Ka , small pKa indicates strong acid ex. pka of NH3 is 9.67 indicates it is a base

Retention of partially folded correct intermediates

related to the free energy The top of the funnel represents denatured conformations—that is, maximal conformational entropy=max energy as protein folds, needs to over come energy barriers, creates intermediates, work on unstable parts of protein until it gets to correct protein, sequential folding (partially folded correct intermediates)

Fed vs. Fasted State

results from changes in blood glucose, liver fed- after meal, glycogenis (production of glycogen), fat and protein synthesis, glucose oxidation fasted- haven;t eaten, glucogenolysis (breakdown of glycogen), gluconeogenisis (using fats and proteins to make glucose), lypolysis, ketonosis (use ketones for energy)

Nonpolar, aromatic amino acids

ring allows them to absorb uv light

difference in states of water

max h bonding in ice, as temp inc h bonds break, as temp cools h bonds form to make ice

Prion protein-

membrane protein found in brain, primarily alpha sheets mutated protein has more beta sheets leads to mad cow disease one diseased structure can convert normal prion protein (transmission)

Chaperonins

protein molecules that assist the proper folding of other proteins, binds to hydrophobic part of aggregate

Ribonuclease

what determines how proteins fold? RNase- Small, heat stable proten ,enzymatic activity, Single polypeptide chain (tertiary structure) with 4 disulfide bonds between 8 cystines in pairs, beta sheets and alpha helixes

Polar (hydrophilic)

• uncharged • Polar, acidic (negative charge, COO-) • Polar, basic (positive charge, NH3 + )

polar covalent bonds

•Electrons not equally shared between atoms •Significance of polarity: relatively reactive

Peptide bonds trans

Peptide bonds exist is trans configuration Avoids steric hindrance between R-groups

Hydrophobic effect

"Bury" the hydrophobic side chains, minimizing contact with water Most polar residues face the outside of the protein and interact with solvent

acids and bases

Acid Proton Donor, Base Proton Acceptor Acids ionize (dissociate) to form a proton and a conj base extent of ionization determines acid strength, expressed as the Ka

General Structure of an Amino Acid at pH 7.0

All amino acids have a carboxyl (--COOH) and amino group (--NH2 ) At pH 7.0, these groups acquire a charge side chain, variable in all 20 amino acids, important for non covalent interactions like peptide bods COO deprotonated NH3 protonated

Disruption of noncovalent interactions on binding oxygen

As O2 binds to Fe, F-helix moves • Loss of interactions between F and H helices. 1. Salt bridge between Asp94 (negative r group) and His146 is broken. (positive r group) 2. Salt bridge between Lys40 (positive r group) and His146 (negative coo-) is disrupted. 3. H-bond between Val98 and Tyr145 also disrupted when o2 binds 15 degree of rotaion occurs, breaks 3 noncovalent bonds, central cavity colapses in oxygenaed hemoglobin

The Bohr Effect: Chemistry of [CO2 ] binding

At low pH and high CO2 in peripheral tissues, Hb affinity for O2 decreases. • CO2 binds to α-amino group at amino terminus of each globin chain— - Amino terminus becomes an anion - reacts with Arg141 of -chain; forms a salt bridge - Deoxygenated (T) state stabilized and promotes release of O2 Hb binds to excess CO2 (and H+);Hb curve shifted to "right", less saturated and better at dropping off o2 at tissues when they are exerting themselves (acidic)

rule of thumb

At pH < pKa , H+ on, protonated At pH > pKa , H+ off, deprotonated

Electrostatic, ionic interactions

Attraction/repulsion of charges Occur between atoms with a complete (-) charge and a complete (+) charge, unlike water partial charge Strength of interaction determined by distance and solvent Would ionic interactions in NaCl be stronger if the salt were dissolved in water or butanol? butanol is nonpolar so na cl wont dissolve in it so attraction between na and cl will be much greater than in water where it dissolves Importance: protein-protein interactions, DNAprotein interactions (sugar phosphate backbone), catalytic mechanisms

Van der Waals forces

Basis: transient asymmetry in the electron distribution of one molecule will induce complementary asymmetry in a nearby molecule, dipole forces Result: charge fluctuations--Transiently produces (+) or (-) poles even in nonpolar molecule Distance matters: present with "snug fit" between atoms Weakest of noncovalent interactions (0.1-1 kcal/mol)

pH homeostasis

Blood pH must be maintained in a narrow range, typically 7.36-7.44 Intracellular pH: 7.1 (range 6.9-7.4)) if pH changes can interrupt breathing

How are misfolded proteins removed from the cell?

Chaperonins- to accelerate slow steps using ATP, unravel a misfolded protein and refold; prevent aggregate formation Chaperonins were first identified as "heatshock proteins" Proteasomes: Degrade proteins to free amino acids if both these systems overwhelmed lots of aggregate formations, amyloid exposed

Hemoglobin Shows Allosteric Behavior

Characteristics of proteins with allosteric behavior - Quaternary structure; different conformational states, R (oxy) and T (deoxy) - Cooperativity leads to change in conformation - Sigmoidal binding curve or kinetics Myoglobin has higher O2 affinity; stores O2 while Hb gives off o2; gives off o2 in high exerting musclesmyoglobin has only 1 subunit so it doesn't work cooperatively, only low around 10 when muscles exert themselvesmyoglobin not circulating protein

Biochemistry

Chemistry of living things interactions and reactions

Ribonuclease experiment

Control: Measure RNAse actiivty 1. Treat pure RNase with urea and excess βmercaptoethanol, strong reducing agent 2. 8M urea is a denaturing agent-breaks all noncovalent interactions, if di sulfide bonds remained after urea signals that protein hasn't completely unfolded 3. β-mercaptoethanol disrupts disulfide bonds 4. These reagents together completely unfold the structure; peptide bonds remain intact. Does it have activity? we predict that if it has been unfolded to primary structure it would have no activity, found this true Would it be able to refold if β-mercaptoethanol and urea removed by dialysis? RNase did regain activity but not 100% assumed some molecules didn't regain, believed specific cystines needed to bind together then adds trace amounts of β-mercaptoethanol, protein refolds, this broke the sulfide bonds that weren't paired correctly and saved the correct ones concluded: RNase can refold by itself solely based on primary structure, amino acid sequence determines how protein folds, need to form correct disulfide bonds, steric and hydrophobic interactions contribute but aren't determining factor

titration curve

Each point on the curve represents changes in ratio of dissociated to undissociated weak acid When [HA] = [A-], the acid is 50% dissociated and pH = pKa midpoint= where pH=pka, concentrations are same

non-covalent interactions

Hydrogen bonds Electrostatic, ionic interactions Van der Waals forces Hydrophobic interactions 1) Weak and transient--gives macromolecules flexibility 2) Provide stability, large number present unlikely they will be broken at the same time. 3) Essential to specificity and catalytic efficiency of enzymes (biological catalysts)

equilibrium constant

If [product] = [reactant] then Keq = 1 and reaction is at equilibrium. rate constants for forward and reverse reactions are equal. no net change over time. If Keq > 1, then [product] > [reactant] means forward reaction is favored

Doxorubicin

Ionizable primary amine pH= 8.2 weak base ions cannot pass cell membrane so what percentage of drug is being ionized? ie. not getting into cell 5% will get into cell will normal cells or cancer cells take up greater amount of drug? More of the drug enters the normal cells vs. cancer cell: 17% vs 5% normal cells can be compromised

What causes a protein to misfold

Mutation-not responsible for misfolding in all cases ex. sickle cell- single amino acid substitution in the beta-chains of hemoglobin: valine replaces glutamic acid, aggregation of Hb molecules and the formation of insoluble fibers that result in the sickle shape Exposure of amyloid segments—Short sequences of ~6 amino acids; present in a large percentage of proteins Normally buried within interior of structure but can become exposed sticky, results in aggregation and the formation of insoluble fibers

Hemoglobin vs myoglobin structure

Myoglobin- stores oxygen in muscle (affects bicarbonate concentration), polypeptide chain; monomer Mb: 153 aa, 17,200 MW small, tertiary hemoglobin-transports oxygen, carbon dioxide, H+ • Important role in pH balance , tetramer: two alpha subunits of 141 residues, 2 beta subunits of 146 residues, quaternary structure no beta sheets in either, all alpha helical, can see heme prosthetics which are bound in the structure, in hemeoglobin there is a cavity without o2 bound

Why is it important to know the classification and side chains/R-groups of the amino acids?

Noncovalent interactions contribute to protein folding and reactivity. Consider the effect of mutations which result in amino acid substitutions: Conserved versus Non-conserved substitution

Cancer cells vs normal cells

Normal differentiated cells=IC pH ~ 7.2,EC pH ~7.4 Cancer cells =IC pH ≥ 7.4,EC pH ~6.7-7.1 cancer cells have reverse gradient, more basic inside and more acidic outside, becaue cancer cells go through glycolysis without oxygen and produce lactic acid allows cancer cells Proliferation, Avoid apoptosis, Migration & invasion, Avoid immune detection

What percent of free Histidine is ionized at pH 7.3?

Only need to consider the R-group since carboxyl group is fully deprotonated and amino group is fully protonated at pH 7.3 use henderson hasslebach Therefore 20 parts [His] in 21 parts of total ([His] + [His+ ]) This means that 95% is [His] and [5%] is [His+ ] histadimine is predominantly uncharged passed 7.3

shared properties of living organisms

Organization and complexity- cells, tissue, starts with water Adaptation- response to environment changes ex. regulation of blood glucose Chemical/energy transformation- organisms are open system, transfer energy with environment, breaking chemical bond energy of c bonds allows synthesis of ATP self-replication- reproduction

Why Heme for Oxygen Binding?

Oxygen is poorly soluble in water. - diffusion through tissue ineffective, needs to bind to a protein Transition metals have a strong tendency to bind oxygen. - very reactive in free form, especially iron. Iron must be sequestered to render it less reactive

Peptide bond resonance

Partial double bond character between oxygen and nitrogen on COO- carbon to maintain planarity Result: Protein backbone rotations are similar to sheets of paper, •free rotation does not occur •rigid planar

Buffers important for cell homeostasis

Phosphate buffer H2PO4 --> H+ + HPO4 -2 pKa = 7.2 intracellular fluids (blood) Carbonic acid / bicarbonate pKa = 6.1 (blood) • CO2 (dissolved) + H2O <--->H2CO3<----> HCO3 - + H+ Carbonic acid/bicarb system works because of high [CO2] dissolved in body fluids and equilibrium between dissolved CO2 and CO2 in lungs. Proteins (amino acids)

hemoglobin and myoglobin

Proteins are evolutionarily related; share sequence homology First proteins to determine structure by X-ray crystallography Both contain the heme prosthetic group- contrubute to function

Reversible ionization

Results from nucleophilic attack by oxygen on a proton of an adjacent water molecule, dissociation doesn't happen as much, forward rxn not favored

What you need to know about the side chains (R-groups)

See handout on HuskyCT memorize side chains of amino acids

Ramachandran Plot

Shows favorable phi-psi angle combinations. 3 main "wells" for α-helices, ß-sheets, and left-handed α-helices. steric effects limit rotation combinations of phi and psi angles

Fetal vs. Adult Hb

Structural difference in HbFHbF curve shifted to "left"Fetal Hb "wins" tug-of-war for o2 since fetus doesn't have lungs, high afinity harder to release Fetal Hb differs from adult Hb: • 2 alpha chains & 2 gamma chains instead of beta • Serine substitutes for His143 in fetal Hb, serine is polar uncharged so serine binds o2 more tightly

Amino acids are optically active

The -carbon is the chiral center of the molecule Exception: Glycine, has chiral H Only the L- configuration is found in proteins

Hydrophobic Effect

The clustering of hydrophobic molecules in water, they are driven to act by entropy when non polar molecules interact, increase in the entropy(confusion) of water, water becomes less ordered, also inc entropy=inc resonance important for cell membrane, phospholipid bi layer with polar head nonpolar tail, amphipathic Hydrophobic Effect important for how proteins fold

Hemoglobin Binds Oxygen Cooperatively

The transition from deoxyhemoglobin (T state) to oxyhemoglobin (R state) occurs upon oxygen binding. • The iron ion moves into the plane of the heme when oxygen binds. The proximal histidine, which is a component of an α helix, moves with the iron. • The resulting structural change is communicated to the other subunits so that the two αβ dimers rotate with respect to each another, resulting in the formation of the R state. one change is subunit causes change in all other subunits

what makes water essential

Water as solvent for reactions and interactions Typical eukaryotic cell: 65-70% water Human body: 65% water Water as a reactant or product in reactions Water as an organizing principle polarity of o, and reverse ionization

Why are many salts soluble in water?

Water has high dielectric constant (80 vs. 1 for a vacuum) What does the dielectric constant tell you? High value indicates greater ability to dissolve salts ex. a lot of drugs made as salts so they can dissolve in blood and be take up

How does the Henderson-Hasselbalch equation explain buffers?

When [HA] = [A-], the acid is 50% dissociated and pH = pKa This is a point of maximum buffering capacity region of buffering capacity is +1 and -1 pH from this point Slope inversely related to buffering capacity, only ratio of acid to base changing

Nonpolar (hydrophobic):

aliphatic- one branch aromatic- 6 member ring

peptide chain (or protein)

amino acid sequence (primary structure) is always written from the amino terminal to the carboxyl terminal, or left to right, zig zag shape of backbone, howproteinsynthesis occurs left to right amino terminal end is beginning carboxyl terminal end is the end only time you see alpha c and alpha amine in repective charged states (nh3, coo-) is at terminal co, carbonyl can engage in h bonding amind, nh can engage in peptide bond

what if If the R group ionizes

amino acid will have a third pKa acids—tyrosine, cysteine, arginine, lysine, histidine, and aspartic and glutamic acids—have readily ionizable side chains

what molecules can dissolve in water

amino acids and glucose cause they can make h bonds with water, fatty acid can't no free electronegative element

Ionization of amino acids

amino acids at least have 2 pka *At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form at pH2= 50% coo protonated 50% deprotonated *At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. the net charge is zero; such ions are called zwitterions at pH9= 50% nh3 protonated 50% nh2 deprotonated *at alkaline pH, the amino group is neutral -NH2 and the amino acid is in the anionic form net charge goes from +1 to 0 to -1

anabolism vs catabolism

anabolism- the process of building up larger molecules from smaller ones, requires energy. ex. synthesis of atp catobolism- Metabolic pathways that break down molecules, releasing energy. ex. break down carbs for atp

hydrogen bonds

between electronegative atom (acceptor) and an H atom that is covalently bonded to another atom (donor) Weak association: ~ 1 to 5 kcal/mole vs. 50 to 200 kcal/mole for covalent bond Rapidly form, break, reform spontaneously Importance: solvent properties of water; structure of proteins and nucleic acids (DNA & RNA), if bonds were covalent for dna when the bonds break during replication large energy released which cell couldn't use but cell can use energy from breaking h bonds, heat also allows h bonds to break in proteins

what can't be stated about proteins

can be determined that amino acid sequence determines folding can't be determined that protein fold by itself if protein is large and complex it needs assistance

molecule structures

can be easily changed by reactions and interactions ex. structure of hemoglobin changes when o2 is bound or not, can also be changed by mutation affecting one amino acid in chain causes sickle cell

when do side chains titrate?

depends on pka of side chain ex. pka of arginine side chain at 12.5 so first coo, then nh3, then side chain where as in histidine its the second one

effectiveness of a buffer

determined by •pK a relative to the pH of the solution •Concentration of buffer components

Amino acid sequence (primary structure)

determines protein folding • Satisfy constraints: • phi and psi angles, -- minimize steric effects • Correct pairing of cysteines for disulfide bonds

Transmission of Misfolded α-Synuclein Between Cells in Parkinson's Disease

diseased protein can be transmitted to healthy cells through exosomes, endo/exocytosis, nono tubles, dying cells if transmission didn't occur unhelthy cell would just die but intead neurodegenerative disease transmits all over body

GERD

gastroesophageal reflux disease, stomach acid refluxes into the esophagus, malfunction of sphincter disease

polar charged amino acids

glutamate, aspartate=Polar charged, negative, acidic lysine, arginine, histadine=Polar charged, positive, basic

Hydrophobic pocket

hb and mb must be able to release o2 fre heme w/o pocket can't release, because iron is reduced to fe3 and o2 becomes oxidized pocket prevents reduction and oxidation

Example:Titration curve and pKa of histidine R-group

imidazole group can be protonated or deprotonated n or nh, acts as acid or base Histidine is found at the active sites of many enzymes that require a proton donor or proton acceptor first group to titrate always alpha carbon cooh group, histidine has +2 net charge pH= 1.8 next to titrate is imidazole group, coo deprotonated, has net charge of +1 pH= 1.8-6 amino group last to titrate, imidazole and coo group deprotonated net charge is 0 pH= 6-9.3 above 9.3 nh2, coo, imadozole, deprotonated

misfolding

incorrect structure, may or may not lose activity Missfolded proteins can: Refold Degraded to free amino acids Forms aggregates or amyloid fibers—leads to disease

pH Low Insertion Peptides (pHLIPs) & Tissue Targeting

more effective way to target disease tissue takes advantage of reverse gradient when these pHLIPs react with the acidic extracellular fluid of the cancer cell they begin to fold and insert into the membrane creating a helix (NH3+ on outside) and can serve as delivery agents couple a drug to COO- side of pHLIP with cleaving enzymes, can release drug to inside cell or couple fluorescence to NH3+ side on outside allows for staining

Sickle cell disease

mutation resulting in the substitution of valine for glutamate at position 6 of the β chains. - Sickle-cell anemia can be fatal when both alleles of the β chain are mutated. - In sickle-cell trait, one allele is mutated and one is normal. Such individuals are asymptomatic

elements in living things

oxygen, hydrogen, and carbon—make up 98% of the atoms in any organism h and o in water, water is essential for life c in key atom of bio molecules

small ph changes in cell

pH increase of 0.2 to 0.3 units promotes cell proliferation, migration, assembly of actin filaments. changes in pH regulate specific proteins (pH sensors) change in binding affinity Ex: ion channels, pumps, enzymes Association with disease ↑pHi -cancer ↓ pHi - several neurodegenerative diseases

Buffering Capacity of Hb

pKa of H2CO3 = 6.35; blood pH = 7.4 Carbonic acid (H2CO3) is the major metabolic acid produced in the body; also a major buffer. Ability to serve as buffer due to dissolved CO2 in body fluids which is 500X > than H2CO3 . Dissolved CO2 is in equilibrium with air in lungs; availability ↑ or ↓ by rate of breathing. co2 promotes release of o2 dec pH and forms carbanate and release of protons

Phi and Psi Bond Angles

peptide bond between c=o and n-h next carbon is alpha carbon, peptide bond can rotate but phi psi can rotate 180 phi= bond between amine and alpha carbon psi= bond between alpha c and carbonyl

2,3-Bisphosphoglycerate (2,3-BPG)

polar charged 2 phosphates related to an intermediate in glycolysis produced in red blood cells w/o it hemoglobin is more hyperbolic 2,3-BPG binds in cavity of hb between two beta subunits his 2, 143 lysine 82 binds by electrostatic forces of these basic amino acids when o2 binds 2,3-BPG released from cell o2 released 2,3-BPG goes in stabalizes t state

Ka acid dissociation constant

strong acids completely dissociate equilibrium constant for dissociation constant for any given acid Ka is large, indicates strong acid, determines which functional group dissociates first proximity of functional groups to functional groups that can act as acid or base affects ka ex. cooh near nh3+ group acid near base, cooh near nh2- will dissociate h from cooh to become coo- and nh3-

Bohr Effect

the regulation of oxygen release from Hb by H+ and CO2 Hemoglobin as a pH Sensor Hb carries 2 end products of respiration spontaneous but dissolved CO2 reacts slowly with H2O to form H2CO3 . Rate is increased by carbonic anhydrase when pH drops from 7.4 to 7.2 release shifts to 77% instead of 66% pH 7.2 and co2 get 88% release, inc co2 promotes release of o2 Protons react with Histidine side chains. His-146 in B-subunit important; protonation promotes release of O2. Acid-base properties of Hb, reform salt bridge with asp 94 pKa of His146 = 6.6 for oxygenated Hb; pKa of His146 = 8.2 in deoxygenated Hb indicates when o2 released and proton bids to imidazole of his 146, his accepts proton acts as base has higher pKa

polar uncharged amino acids

tyrosine can form h bonds

Denaturation

unfolded Loss of secondary and higher order structure Noncovalent interactions are disrupted; peptide bonds are not, loss of activity Example: Ribonuclease Expt (agents), extreme pH, detergents, heat