Lab Exam

Question 1

properties of enzymes

Answer 1

bind reactant (Substrate), alter its configuration so that it is more easily changed into the product, enzyme remains unchanged over the course of the reaction

Question 2

enzyme and Ea

Answer 2

Ea: activation energy - energy barrier needed to overcome for a cellular reaction to occur

enzymes reduce the Ea for the reaction, and increase RR/velocities of the reaction.

• do not affect ∆G’º
• decrease Ea/∆G†
• speed up reversible reactions by the same degree in both directions

draw diagram

Question 3

Michaelis-Mentin plot

Answer 3

define relationship between [S], V0 and Vmax
contestant Km = [S] when 1/2Vmax = V0
draw diagram

Question 4

Km measures what?

Answer 4

measure of the efficiency of an enzyme, Km is the concentration of the substrate which produces a RR of half Vmax

a low Km (closer to zero) indicates high affinity of E for S

Question 5

enzyme kinetics what does each value stand for

Answer 5

V0 = initial velocity or initial RR of enzyme r’n

Vmax: max velocity of enzyme r’n

Km = 1/2Vmax indicates efficiency of enzyme

Question 6

steady state kinetics

Answer 6

kinetics of enzyme r’n after first few microseconds when [ES] and conc of any other intermediates remain constant,
usually applies to measurement of V0

Question 7

lineweaver-burk plot (Double reciprocal)

Answer 7

y axis: 1/V0
x axis 1/[S]

y - axis intercept: 1/Vmax
x - axis intercept: -1/Km

can use y = mx + c to find values of graph, eg x = 0 to find y int

Question 8

factors affecting protein structure

denaturation vs renaturation

Answer 8

pH, temp, solubility, ionic strength

den: loss of biological activity
ren: regains biological activity

Question 9

enzyme inhibition

Answer 9

inhibitors: compounds that decrease enzymes activity

Question 10

irreversible inhibitor

Answer 10

inactivator that reacts with the enzyme, permanently shut off the enzyme molecule.
often toxins, can be used as drugs

Question 11

reversible inhibitor

2 ways of functioning

Answer 11

bind and dissociate from an enzyme, temporary inhibiton

often structural analogy of substrate or product, used as drugs to slow down a specific enzyme

1. bind to free enzyme and prevent substrate from binding - temporarily, eventually dissolve and enzyme works again
2. bind to the ES-complex and prevent reaction from occurring (Temporarily)

Question 12

competitive inhibition

Answer 12

lines intersect on the y-axis
∆ 1/Km, same 1/Vmax
binds directly to AS, competing directly with substrate

Question 13

uncompetitive inhibition

Answer 13

lines parallel across y-axis
dec 1/Km and dec 1/Vmax

binds to another part of enzyme (not AS) sits on allosteric site, allows substrate to bind but interferes with efficiency and RR

Question 14

mixed inhibition

Answer 14

lines intersect before the y-axis
inc 1/Km, dec 1/Vmax
most effective, bind to AS, allosteric site, substrate or anything on enzyme and shut it off, decreases efficiency of enzyme and interferes with velocity / RR

Question 15

noncompetitive inhibition

Answer 15

lines start together on -x-axis
same 1/Km, dec 1/Vmax
bind to allosteric site, doesn’t interfere with enzyme binding to substrate, interferes with velocity, and stops overall reaction

Question 16

Bradfords assay:

Answer 16

shifts absorbance from 470nm to 595nm
intensity of blue correlates with concentration of proteins,
measured qualitatively with eye (compare colours) or quantitatively with spectrophometer

dye: Coomassie brilliant blue dye

Question 17

limitations of Bradfords assay

Answer 17

measures total [protein], need further methods to identify specific proteins
assay is linear over limited range
dye binds directly to arginine and hydrophobic AA
AA composition can alter the concentration-absorbance curve
standard with a similar composition to unknown must be used - eg BSA

Question 18

Beer’s Law

Answer 18

states that if a solute absorbs light of a particular wavelength, the absorbance is directly proportional to the concentration of that solute in solution up to a point

Question 19

Beer’s law formula

Answer 19

A = kLc / A = Ebc
k - constant / molar absorptivity
L - light path length (~1cm)
c - concentration of compound in solution

Question 20

qualitative vs quantitative determination of protein concentration

Answer 20

qualitative: visually compare the colour of unknown sample against standards of known concentration
quantitative: A595nm for each standard and generate standard curve with data, unknown sample into the linear equation to determine concentration

Question 21

properties proteins can be separated by:

Answer 21

charge, size, affinity for a ligand, solubility, hydrophobicity, thermal stability

chromatography used for protein separation based on one of those properties

Question 22

chromatography

Answer 22

distribution of compounds between 2 immiscible phases
- stationary phase and mobile phase
selection of the 2 immiscible phases is dependent on the characteristics of the molecules of interest

Question 23

size exclusion chromatography

Answer 23

separation of proteins based on molecular size. large molecules too big for the bead pores eluted first, smaller molecules interact with bead pores and so elute later.

elution time/volume is inversely proportional to molecular weight (molecular weight not proportional to molecular size)

Question 24

ion exchange chromatography

Answer 24

separation by charge. proteins charged due to phosphate group and electrostatically bind to opposite charges.

columns covalently bond charged molecules to the insoluble resin. eg with a cation exchanger, proteins with more negative net change move faster and elute earlier

Question 25

ligand affinity chromatography

Answer 25

solution of ligand is added to column, specific to protein of interest. protein of interest eluted first by ligand solution.

Question 26

electrophoresis

Answer 26

analytical separation of proteins. electrical field pulls/separates proteins according to charge. travel towards +ve terminal. gel matrix hinders mobility of proteins according to size and shape - eg only separated out by charge.

Question 27

SDS-PAGE

Answer 27

used to calculate molecular weight of protein

sodium dodecyl sulfate - polyacrylamide gel electrophoresis

Question 28

plasmids 3 essential parts

Answer 28

plasmid: circular piece of bacterial DNA
ori: origin of replication. DNA polymerase/other proteins required for DNA synthesis bind to the ori

Selectable marker: usually drug resistance. provides a way to select for bacteria that contain plasmid

MCS: multiple cloning site (not essential), region with several restriction enzyme recognition sequins the provide cloning sites for insertion of your gene of interest. MCS used to cut open because lots of palindromes are present
- 2nd fail safe: LacZ operon, gives bacteria availability to break down lactose into galactose and glucose - in the event that the B-galactosidase gene is disrupted

Question 29

palindrome defn and fn

Answer 29

sequence of DNA that is the same when read in opposite directions (fwd and back) eg GAATTC is CTTAAG

Question 30

restriction enzymes

Answer 30

specific endonuclease (cut inside DNA, as plasmids are circular, no need for exonuclease)

• recognise short sequence of NDA and cleave DNA at or near the recognition sequence (palindromes)
• cut into sticky or blunt ends (sticky ends can bind with other DNA molecules with same overhands).

serve as a natural defence mechanism for bacteria against viral infection, prevent replication if phage DNA by cutting it into too many pieces

Question 31

DNA ligase fn

Answer 31

enzyme that joints the ends of DNA and re-establishes the phosphidester bond in the DNA molecule, puts sticky and blunt ends back together