BioChem 501 Unit 1 Exam Review

size of bacterial cell and animal cell (micrometers)

1, 50

important trace elements

iron, copper, zinc

enthalpy, H (units) +/-

amount of energy in a chemical bond (J/cal), - heat released

entropy, S (units) +/-

randomness (J/K), + more disorder

hydrophobic effect (thermodynamically favorable?)

ordering of water molecules around a hydrophobic molecules, reduces entropy: unfavorable

how do hydrophobic molecules arrange themselves in water?

into micelles, essentially large clusters with hydrophobic interior and hydrophilic exteriors, increases entropy: favorable

acid

proton donor, electron pair acceptor

base

proton acceptor, electron pair donor

Henderson-Hasselbach equation

pH = pKa + log[A-]/[HA]

do an ICE table (or don't)

how'd it go?

important buffers in blood

phosphate, bicarbonate

are amino acids D or L stereoisomers?

L

typical cell contains ____-____ proteins

8,000-10,000

functional group of a peptide bond

amide

post-translational modifications (2 examples)

add chemical diversity to proteins, enzymes recognize peptide sequences on proteins, covalently bond, disulfide bridges, phosphorylation

steps of protein purification (3)

choose protein source, disrupt cell to solubilize protein (grinding, sonication, pressure, osmotic shock), purification

protein purification strategies based on properties (3)

charge: ion exchange chromatography, isoelectric focusing; size: gel electrophoresis, gel filtration/size exclusion chromatography, specificity: affinity chromatography

amino acid that absorbs UV light efficiently (wavelength)

tryptophan, ~280 nm

ion exchange chromatography

positively charged proteins stick to negatively charged beads, elution (removal) of protein achieved by changing salt conditions: add NaCl to neutralize charges, first proteins to come out: most negative

size exclusion chromatography/gel filtration

porous column acts at molecular sieve, smaller molecules get stuck in pores and large proteins pass through

affinity chromatography

protein isolated by binding to a ligand, elution achieved with high concentration of free ligand, unwanted proteins washed through first

specific activity (units/mg)

higher value: more purity

electrophoresis

separation on the basis of charge by application of an electric field, or size when denatured by detergent (SDS)

isoelectric focusing

useful if proteins have same molecular mass but different charges

mass spectrometry

measures mass, gel molecules fly in gas phase by electrospray ionization, separate ions by mass in a vacuum: lighter ones go farther; can be used to sequence a protein by fragments

properties of peptide bonds (2)

planar, small electric dipole

Ramachandran plot

shows allowed regions of protein folding space

properties of alpha helices (4) (handedness?)

right handed, 3.6 AA/turn, H-bond: carbonyl n/N-H n+4, side chains protrude out

properties of beta sheets (3)

made of beta strands, 2 types: parallel/antiparallel, strands contain few AA (3-10)

antiparallel beta sheets

H-bonds parallel to sheets, backbones alternating

parallel beta sheets

peptide backbone in same direction, H-bonds at weakened angles

fibrous proteins (3)

repeating helices/sheets, abundant proteins (structural), keratin/collagen

keratin

hair, skin, feathers, nails; helices linked by disulfide bonds, high tensile strength

collagen

bone, cartilage, connective tissue; triple helix of polymer (Gly,Pro,HyPro), high tensile strength

protein stability (define)

difference in free energy between folded and unfolded state, ~-20kcal/mol; comes from hydrophobic effect

benefits to larger proteins (2)

more efficient: only have to code smaller segments, error rate 1/10,000 AA

Anfinsen's experiment

AA sequence contains info required to fold into 3D structure

myoglobin (3)

oxygen storage protein (NOT transporter: binds O2 too tightly), abundant in mammalian muscle tissue esp. diving animals, 153 AA (small): 8 alpha helices (monomer)

heme group (synthesis, structure, 2+/3+)

synthesized in red blood cells, Fe inside a porphyrin ring, 2+ state binds O2 and 3+ state does not

oxygen binding site in myoglobin

forms ~120* bond to Fe and partial H-bond to distal histidine

CO binding to heme

20,000x stronger to free heme than O2, only 200x stronger to myoglobin (H-bonding), can treat quickly with pure O2

hemoglobin (3)

oxygen transporter in blood, tetramer: alpha/beta subunits, 4 hemes (binds 4 O's)

T and R states of hemoglobin (O, other ligands, stability, venous/arterial)

oxygen binds to both but stronger to R, stabilized R state, T-venous and R-arterial, CO2 also a ligand

hemoglobin and myoglobin oxygen binding curves

myo: hyperbolic (hugs axes), hemo: sigmoidal (result of cooperativity),

cooperativity of oxygen binding in hemoglobin (3)

when first oxygen binds it does so loosely, alters conformations of other three subunits toward R-state, following O's bind more tightly

hemoglobin also transports ___ and ___ from tissues to lungs (cellular respiration by-products), location?

CO2 (terminal amino groups) and protons (side chains with altered pKa's) during T state

Bohr effect

low pH stabilizes T state, high pH stabilizes R

2,3-bisphosphoglycerate (5)

regulates binding affinity of hemoglobin for oxygen (stabilizes T state), without it binding curve is hyperbolic, allows cooperativity, more BPG: release more O2 to tissues, BPG binds in cavity between subunits (one per tetramer)

hemoglobin's binding sites (4)

O2, CO2, H+, BPG

enzyme classification (6)

oxidoreductase (redox), transferase (methylation/phosphorylation), hydrolase (hydrolysis), lyase (addition of groups to double bonds), isomerase (transfer of groups to yield isomers), ligase (joining two molecules/hydrolysis of ATP)

what state is the enzyme complementary to?

transition state (E-S complex)

catalytic strategies of enzymes (3)

general acid-base catalysis, covalent catalysis, metal ion catalysis (can use >1 at same time)

acid-base catalysis

enzyme provides additional functional groups

covalent catalysis (example)

formation of covalent bonds to enzyme, e.g. serine proteases (digestive)

metal ion catalysis (5 examples)

1/3 of all enzymes use, stabilize charge buildup at TS, e.g. Ca2+, Zn2+, Mg, Mn, Zinc proteases

rate determining step of an enzyme-driven reaction

ES to E and P

define/describe Km

1/2Vmax, indication of how tightly the enzyme binds substrate

kcat

first order rate constant, "turnover number" #/s

kcat/km

catalytic efficiency, most efficient: match rate of diffusion (10^8/Ms)

chymotrypsin

protease that digests proteins containing aromatic AA residues, produced in pancreas and injected into small intestine

chymotrypsin mechanism (7 steps) [you can do this!!!]

(1) substrate binds (R-group) into hydrophobic pocket, (2) alkoxide ion attacks carbonyl C forming a tetrahedral acyl-enzyme (O- stabilized by oxyanion hole), (3) peptide bond breaks as carbonyl reforms and first product is released, (4) water deprotonated and OH- attacks ester bond (O- stabilized by hole), (5) formation of second transition state stabilized by oxyanion hole, (6) collapse of tetrahedral intermediate forms second product (carboxylate anion), (7) product 2 is released and active site regenerates free enzyme

important features of chymotrypsin active site (4)

Ser195: acylation/covalent chemistry, His57: general acid-base, oxyanion hole: stabilizes TS, hydrophobic pocket: R-group ring binds

regulatory enzymes (3)

allosteric enzymes, covalently modified enzymes, zymogens

allosteric enzymes (example)

bind regulatory compounds (modulators) non-covalently (reversibly), e.g. aspartate transcarbamoylase

covalently modified enzymes (example)

regulatory compounds covalently attached in reversible manner, e.g. post-translational modification (glycogen phosphorylase)

zymogens (example)

enzymes made as inactive precursors that need to be cleaved to become active, e.g. chymotrypsinogen

reversible inhibitor characteristics (3 examples)

generally small molecules that bind in/close to active site, competitive/uncompetitive/mixed

irreversible inhibitors (5 examples)

covalently attach to enzyme, "suicide substrates", basis of many pharmaceutical agents, e.g. aspirin, diisopropylfluorophosphate, inactivates trypsin/chymotrypsin/acetylcholinesterase

competitive inhibition (Vmax/Km/slope)

Vmax: no change, Km: increase, increasing slope on graph

uncompetitive inhibition (Vmax/Km/slope)

Vmax: decrease, Km: decrease, same slope on graph; inhibitor binds after substrate does

mixed inhibition (Vmax/Km/slope)

Vmax: decrease, Km: increase, increasing slope on graph

lipid functions (7)

principle energy stores, membranes, cofactors, electron carriers, pigments, hormones, messengers

oleic and elaidic acid (cis/trans)

oleic: cis, elaidic: trans

triacylglycerols are efficient energy sources because... (3)

highly reduced (lots of e-), provide >2x energy as carbs, dehydrated: pack together through hydrophobic effect; disadvantage: metabolized more slowly

structural lipids (3)

glycerophospholipids, sphingolipids, sterols

glycerophospholipids

glycerol 3-phosphate backbone, ester bonds to 2 fatty acids

sphingolipids

sphingosine backbone (15-C chain attached to glycerol), amide bond to 1 fatty acid, important immunogenic determinants in blood (head group: blood type)

glycerophospholipids in cellular signalling (think products of cleavage)

prostaglandins are derivatives of membrane GPL containing 20-C arachidonic acid, responses: inflammation, pain/fever, BP, clotting, reproductive function, sleep/wake cycle

sterol structure (5 examples)

mainly cholesterol: hydrophobic fused rings, alkyl tail, polar head group; also bile acids, testosterone, estradiol, cortisol, aldosterone

lipid bilayer thickness (# AA)

3nm, 20-25 AA

bilayers wrap around to form continuous, spherical particles called _____, energy required?

liposomes, no: form spontaneously

diffusion of leaflets

transbilayer: t1/2 in days, lateral: 1 micrometer/s

disrupting membrane proteins

peripheral: change pH (salt concentration) by chelating agent/urea/carbonate, integral: add detergent

hydrophobicity profile

~20-25 AA groupings of hydrophobic R-groups above x-axis, hydrophilic (charged) below x-axis, e.g. bacteriorhodopsin (7 hydrophobic helices)

beta-barrel membrane proteins

built from b-strands, hydrophobic parts facing bilayer, hydrophilic line pore and upper/lower outer surfaces, allows selective facilitated diffusion of ions/small molecules

major passive transporters in facilitated diffusion (2 + examples)

channels (e.g. porin, sieves based on MW/charge, may have single gate) and passive transporters (usually 2 gates- 1 in, 1 out, e.g. glucose transporters in erythrocytes)

functions of Na/K pump (4)

transport of Na/K, establish EC gradient, nerve transmission, maintenance of osmotic pressure