BIOCHEM Exam 2

Includes lectures 5-11

Single protein isolation

1. open up cell to extract cell contents

2. centrifugation

3. supernatant in column chromatography

4. collect fractions

5. analyze purity by gel electrophoresis

6. analyze protein characteristics/sequence with protein digestion + edman degradation

what is ion exchange?

A technique used to separate proteins based on their charge by using charged resin in a column.

in ion exchange, (for anions) which is the first to separate?

the least negatively charged (+ → -)

in ion exchange, (for cations) which is the first to separate?

the least positively charged (- → +)

what is gel filtration?

separates proteins by size

for gel filtration, which pass first?

larger molecules because smaller molecules fall into gel pores

what is affinity chromatography?

separates molecules based on interactions w/ stationary and mobile phases

what is gel electrophoresis?

molecules pulled through gel matrix based on protein size

what do proteins need to be treated w/ in gel electrophoresis that DNA doesnt?

SDS-PAGE denatures/unfolds proteins to give them a uniform (-) charge

how does gel electrophoresis work?

(-) molecules loaded in the (-) gel end, with electric field, theyre pulled through to (+) end. larger the molecule, the slower it is

what is trypsin?

an enzyme that cleaves peptide bonds after Lys and Arg

what is chymotrypsin?

enzyme cleaves after Trp, Tyr, Phe

what is cyanogen bromide?

cleaves after Met

what is edman degradation?

a method to determine sequence of short peptides where each AA is cleaved and analyzed until all AA is identified

what do catalysts do?

increase rxn rate w/o being chemically modified

what is activation energy and its symbol?

ΔG゜‡, energy needed to reach transition state

what is free energy change and its symbol?

ΔG, energy needed to change between reactants and pdts

what is transition state?

the max point of rxn curve where energy is max

identify each one

how do catalysts/enzymes affect rxn rates?

• lowers ΔG゜‡/activation energy

• increases rxn rate

• doesnt affect transition state or ΔG/free energy change

what is a substrate?

reactant forms complex w/ enzyme

what is a transition state species?

rxn intermediate @ transition state

what is an active site?

site on enzyme for substrate binding

explain lock & key

substrate w/ perfect fit to specific enzyme, doesnt take into account conformational change in enzyme after substrate binding

which model is preferred (lock & key or induced fit)?

induced fit because it takes into account the conformational change

explain induced fit

not perfect fit, but substrate/enzyme changes

how is ΔG゜‡ affected in lock & key?

ΔG゜‡ too high for rxn to happen

how is ΔG゜‡ affected in induced fit?

ΔG゜‡ kept low for rxn to happen

identify each term: E + S → ES → EX‡ → E + P

E- enzyme

S- substrate

ES- enzyme/substrate

EX‡- enzyme/transition state

P- product

What did Michaelis & Menten do?

found kinetics of enzyme-substrate interaction

fill in the blanks for Michaelis-Menten Enzyme Kinetics graph

what type of curve is the Michaelis-Menten Enzyme kinetics graph?

hyperbolic curve

what did Lineweaver & Burk do?

determined how to find Km & Vmax

what type of curve is the Lineweaver-Burk double reciprocal plot?

straight line

fill in the blanks for Lineweaver-Burk double reciprocal plot

where is hexokinase located?

tissues/muscles

what does hexokinase do?

converts glucose → G6P for glycolysis (energy)

where is glucokinase located?

liver

what does glucokinase do?

G6P in liver converted to glycogen to store but broken down when blood glucose levels are low 

specify inactive or active for glucokinase and hexokinase when blood glucose is low

glucokinase- inactive

hexokinase- active

specify inactive or active for glucokinase and hexokinase when blood glucose is high

glucokinase- active

hexokinase- inactive

what do enzyme inhibitors do?

interferes w/ enzyme activity + slows rxn

what do reversible enzyme inhibitors do?

bind to the enzyme, inhibit the rxn, then leave

enzyme is left in original condition

what are competitive inhibitors

competes w/ substrate for active site

what are noncompetitive inhibitors?

they bind at a different site than substrates, cause conformational change for enzyme to become inactive

what do irreversible enzyme inhibitors do?

they bind to enzyme, make it permanently inactive, not released

how do competitive inhibitors effect slope, Km, and Vmax?

• slope - changes

• Km - changes

• Vmax - same

how do noncompetitive inhibitors effect slope, Km, and Vmax?

• slope - changes

• Km - same

• Vmax - changes (decreases)

can competitive inhibitors or noncompetitive inhibitors be overcome with high [S]?

competitive

what is the Michaelis-Menten equation? (enzyme rxn velocity)

V₀ = (V_max [S]) / (K_m + [S])
K_m = [S] when V₀ = ½ V_max

what is the Lineweaver-Burk double reciprocal plot?

1/V₀ = (K_m / V_max) * ((1 / [S]) + (1 / V_max))

in ion exchange chromatography, what charge do you want your stationary phase to be?

opposite to wanted material

what does SDS-PAGE stand for?

sodium dodecyl sulfate - polyacrylamide gel electrophoresis

determine where trypsin cleaves this peptide bond:
Leu - Ser - Tyr - Ala - Ile - Arg - Asp - Gly - Met - Phe - Val - Lys

Leu - Ser - Tyr - Ala - Ile - Arg

Asp - Gly - Met - Phe - Val - Lys

determine where chymotrypsin cleaves this peptide bond:

Asp - Gly - Met - Phe - Val - Lys - Leu - Ser - Tyr - Ala - Ile - Arg

Asp - Gly - Met - Phe

Val - Lys - Leu - Ser - Tyr

Ala - Ile - Arg

what is catalysis?

acceleration of rxn by catalyst

is oxidation of glucose spontaneous or nonspontaneous? ΔG be + or - ? favorable or not?

spontaneous

ΔG = ( - )

favorable

what type of bond forms between enzyme-substrate?

noncovalent

when binding, what part of the enzyme does the substrate interact with?

side chain + backbone groups

what is K_m?

inverse measure of affinity of an enzyme for its substrate

• [S] which 50% of enzyme is occupied by substrate

what is the main concern with Michaelis-Menten Enzyme Kinetics curve?

V_max is asymptotic so impossible to determine K_m at V_max

when solving for Lineweaver-Burk reciprocal plot, what is the equation for y-intercept?

1 / V_max

when solving for Lineweaver-Burk reciprocal plot, what is the equation for x-intercept?

- 1 / K_m

what is phosphorylation?

covalent binding of phosphate group

is phosphorylation reversible?

yes

what AAs can be phosphorylated?

Ser, Thr, Tyr

what is allosteric regulation?

allosteric site on enzyme for effector molecules to bind for conformational change active/inactive

what does kinase do?

enzyme that transfers P group from ATP to other protein/enzyme

what is feedback regulation?

end product of pathway can turn off enzyme @ beginning of pathway

what is zymogen activation?

inactive form of enzyme, cant function until activated usually by cleaving part of its structure

what is a cofactor?

small molecules needed for enzyme activity

what does hemoglobin do?

pick up oxygen in lungs, drops off in tissues

what happens during phosphorylation in Na⁺/K⁺ pump?

causes conformational changes that pumps 3 Na⁺ out of cell

what happens during dephosphorylation in Na⁺/K⁺ pump?

causes conformational changes that pumps 2 K⁺ into cell

what enzyme is involved in CTP production?

ATCase

what kind of curve is the ATCase activity curve?

sigmoidal

what is CTP?

nucleotide base for RNA/DNA synthesis

how does CTP regulate its own synthesis (feedback inhibition)?

• CTP binds to regulatory dimer of enzyme ATCase

• conformational change to reduce enzyme activity

• high amount of CTP end pdt inhibits early step in pathway

• shutdown production + saves energy

• low CTP = slow RNA/DNA synthesis

how does ATP regulate CTP synthesis?

• ATP binds to same regulatory dimer site in ATCase as CTP

• conformational change to increase enzyme activity

• high ATP = accelerates CTP synthesis

how many ATP does the calvin cycle use?

18 ATP

how many ATP does CTP synthesis use?

5 ATP

what are membranes?

lipid layer to separate functions outside/inside cells

what is amphipathic lipid?

2 affinities for water due to polar/hydrophilic head and nonpolar/hydrophobic tail

what are some characteristics of saturated fatty acids?

no C=C

solid at room temp

what are some characteristics of unsaturated fatty acids?

C=C

liquid at room temp

reduces FA melting point

usually conjugated double bond

what is a conjugated double bond?

alternating double and single bonds - more stable

what is a nonconjugated double bond?

double bond separated by 2+ single bonds

FA notation- fill in the blanks

what is hydrogenation?

adding Hs to FA to convert C=C (unsaturated) → C-C (saturated)

what is partial hydrogenation?

not all C=C reduced w/ Hs → C-C

what is complete hydrogenation?

all C=C reduced w/ Hs → C-C

how do nutritionists number FAs?

begin (1) at methyl end → end at carboxyl end

how do biochemists number FAs?

begin (1) at carboxyl end → end at methyl end

characteristics of omega-3 FAs?

• unsaturated

• get from diet: fish, seeds

• health benefits: better blood circulation, lower blood pressure, anti-cancer, anti-depression

what are triglycerides/triacylglycerols?

ester linkage of FAs to each (3) OH glycerols

triglycerides/triacylglycerols function?

stored energy in adipose tissue

what structure is this?

triglyceride/triacylglycerols

are triglycerides completely hydrophobic or hydrophilic?

hydrophobic

what are phosphoglycerides/phosphoacylglycerols?

ester linkage of 2 FAs + PO₄ to glycerol

what is phosphoatidic acid?

phosphoacylglycerols w/o additional group on PO₄