biochem 501 exam 1

why is water denser than ice?

liquid water averages 3.4 hydrogen bonds per H2O molecule while ice has 4

hydrophobic effect

-ordering water molecules around a hydrophobic molecule, reducing the entropy of water and making the reaction thermodynamically unfavorable

-spontaneous clustering of non-polar groups maximizes the entropy of water

clustered lipids release water

entropically favorable

dispersed lipids surrounded by ordered water

entropically unfavorable

why is ionization of water important?

many reactions depend on the proton concentration of H+

acids

proton donors/e pair acceptors

bases

proton acceptors/e pair donors

stronger acids have a ______ tendency to release their protons

greater

two important buffers in blood are…

H₂PO^4- (phosphate) and HCO^3- (bicarbonate)

all proteins are ______ of amino acids

polymers

all amino acids in proteins are _-stereoisomers (except ______)

L, glycine

peptide bond formation is a _____ reaction

condensation

peptide bond breakage is a _____ reaction

hydrolysis

protein purification steps

1. choose protein

2. gently disrupt the cell to solubilize the protein (grinding, pressure, osmotic shock)

3. purification

strategies for purification (charge)

ion exchange chromatography, isoelectric focusing

strategies for purification (size)

gel electrophoresis, size exclusion chromatography

strategies for purification (specificity)

affinity chromatography

tryptophan, tyrosine, phenylalanine & absorbing UV light

tryptophan - yes

tyrosine - inefficient

phenylalanine - no

ion exchange chromatography

positively charges proteins stick to negatively charged beads, elution is achieved by changing salt conditions

size exclusion chromatography

a porous column acts as a molecular sieve that smaller molecules get stuck in pores and take longer to travel through the column

affinity chromatography

protein is isolated by binding to a ligand, elution is achieved with a high concentration of free ligand

protein sequencing uses:

1. identify a protein

2. identify mutations involved in disease

3. understand shape and function of protein

mass spec

lighter ions of molecule goes further

tandem mass spec

can be used to sequence a protein by identifying fragments of unique mass

the two bond angles in a peptide backbone that can rotate

phi and psi

primary structure

amino acid residules

secondary structure

alpha helix

tertiary structure

polypeptide chain

quaternary structure

assembled subunits

alpha helix properties

-right handed

-3.6 amino acid turns

-H bond in carbonyl group

-side chains protrude out from helix

an alpha helix that binds to a negatively charged ion exchange chromatography column is likely to have which amino acids

lysine and arginine (they are positively charged)

beta sheets

-parallel and anti-parallel

-H bonds formed by stands

-side chains on alternate sides form pleated sheet

-sheets twist (NOT flat)

-strands contain few amino acids (3-10)

antiparallel

parallel

fibrous proteins

-highly extended

-repeated helical or beta sheet structure

-abundant in keratin and collagen

keratin

-extended alpha helix, cross-linked by disulfide bonds

-composed of hydrophobic residues

-high tensile strength

collagen

-triple helix of a polymer with a repeating motif (gly, pro, hyp)

-most abundant protein in humans

-high tensile strength

________ is a post-translational modification that is required for collagen to form a stable, coiled structure

hydroxyproline

enzyme that makes hydroxyproline requires ____ as a cofactor

vitamin C

the major source of protein stability

the hydrophobic effect

quaternary structure

-multimers

-hemoglobin for ex.

-enzymes are multimers!

hydrophobic effect

sequestering of hydrophobic side chains into the interior of the protein in a folded state that releases ordered water

2 reasons theres a limit to protein size

1. more efficient to build large structures from smaller ones

2. error rate in protein synthesis is 1 mistake per 10,000 amino acids

myoglobin

oxygen storage protein abundant in mammalian muscle tissue, 153 AA’s, 8 alpha-helices

porphyrin ring in heme

flat, aromatic, provides four nitrogen ligands to the iron to help stabalize

oxygen binds at an angle of ~__ to iron, to the distal histidine

120 degrees

nearly all oxygen is transported bound to hemoglobin in …

erythrocytes (arterial blood = 96% saturated, venous = 64%)

hemoglobin is a _____, while myoglobin is a ______

tetramer (alpha and beta), monomer

oxygen stabilizes the _ state

R

T/F - T state has more interactions between the subunits than the R state, changes occur at the A-B heterodimers

true!

T-state

R-state

oxygen binding moves the _____ histidine, which pulls on helix _, which changes the conformation of the interface

proximal, F

oxygen binding to hemoglobin is….

sigmoidal (result of cooperativity between the subunits)

cooperativity

provides a route towards regulating the affinity of hemoglobin for oxygen through interactions with other ligands, binding of the first allows other molecules to bind more tightly

hemoglobin also transports __ and _ from the tissues to the lungs, removing end products of cellular respiration

CO₂, H⁺

low pH

-favors the uptake of protons and release of O₂

-stabilizes T-state

-binding affinity of hemoglobin for oxygen decreases

__________ regulates the binding affinity of hemoglobin for oxygen, without, there is no cooperativity

2,3-bisphosphoglycerate (BPG)

hemoglobin is an ______ protein: ligand binding induces a conformational change

allosteric

BPG…

-is an allosteric regulator of O₂ binding (lowers affinity)

-increases at high altitudes

-binds in the cavity between the subunits in the T-state (to positively charged side chains, only one BPG binds per tetramer)

enzyme properties

-usually proteins (except when catalytic RNA aka ribozymes)

-good catalysts

-highly specific

-provide control over many metabolic processes

-three dimensional structure of the protein is essential for the activity

enzymes are essential for life because….

they allow biomolecules and metabolites to be synthesized and degraded under physiological conditions where chemical reactions would not proceed at a meaningful rate otherwise

enzymes catalyze reactions by ________ of the transition state

lowering the activation energy

enzymes employ three common catalytic strategies

1. general acid-base catalysis

2. covalent catalysis

3. metal ion catalysis

general acid base catalysis

enzyme provides additional functional groups that aid in catalysis once the substrate is bound

covalent catalysis

characterized by the formation of a covalent bond between the enzyme and substrate, bond must be broken later in the cycle to release the product and regenerate the free enzyme

metal ion catalysis

have positive charges that stabilize negatively charged transition states

enzymes can change the ___ of the reaction but not the ______________

rate, equilibrium constant

rate of reaction is dependent on…..

substrate concentration

enzyme activity is dependent on __ and _____________

pH, temperature

enzyme specificity

small changes in substrate structure that have large effects on enzymatic parameters

chymotrypsin

-a protease that digests proteins containing aromatic amino acid residues

-3 chains held together through disulfide bonds

features of chymotrypsin active site

oxyanion hole: lowers EA by stabilizing oxyanion in TS

hydrophobic pocket: substrate binding and specificity

ser: covalent catalyst

his: general acid-base catalyst

asp: hydrogen bonds to His to stabilize and charge His

regulatory enzymes

must exhibit increased/decreased activity in response to certain signals

allosteric enzymes - quaternary structure w both catalytic and regulatory subunits

bind regulatory compounds (allosteric modulators) reversibly (non-covalently)

covalently modified enzymes

regulatory compounds are covalently attached in a reversible manner

zymogens

enzymes made as inactive precursors that need to be cleaved to become active

allosteric enzymes have non-Michaelis-menten behavior meaning…

they can bind either positive or negative modulators

covalent regulation involves..

post translational modification

feedback inhibition

a product shuts off its own synthesis by negatively regulating an enzyme in the synthesis pathway

ribozyme

active site made entirely of RNA

reversible inhibitors

small molecules that bind in or close to active site

reversible inhibitors type

competitive, uncompetitive, mixed

irreversible inhibitors

covalently attach to enzyme, basis of many pharmaceutical agents

lineweaver burke plot is a ___________ plot

double reciprocal

competitive inhibition does not change ….

V_max

uncompetitive inhibition, … and …. are affected ______

K_M, V_max, equally

mixed inhibition, both …. and …. affected, ______

K_M, V_max, unequally

irreversible inhibition inactives …

trypsin and chymotrypsin, as well as acetycholinesterase

biological lipids

A diverse class of organic molecules that share the common feature of insolubility in water

biological lipids functions

-Principal forms of energy stores in many organisms

-Biological membranes

-Cofactors, electron carriers, pigments, hormones and

messengers

saturated lipids have higher ______ than unsaturated lipids

melting points

lipids from fatty acids

-Triacylglycerols are fatty acids attached to glycerol through ester linkages.

-Principal component of fat cells (adipocytes) and seeds

-Long-term energy storage

Triacylglycerols are efficient source of energy because

1. They are highly reduced

2. Provide >2 x the energy as carbohydrates

3. They are dehydrated and hence take up less space (and weight) than the equivalent carbohydrate

Disadvantage of Triacylglycerols as an energy source

They are metabolized more slowly than glycogen, starch, or other carbohydrates

There are three major classes of lipids found in membranes

I. Glycerophospholipids

II. Sphingolipids

III. Sterols

Glycerophospholipids

Glycerol 3-phosphate: The backbone of glycerophospholipids

Sphingolipids

sphingosine backbone instead of glycerol 3-P, important immunogenic determinants in blood