Enzyme function

Question 1

Control of enzyme activity

Answer 1

gene expression, protein degradation, covalent modification (phosphorylation), allosteric control, protein binding, zymogens

Question 2

Cooperativity

Answer 2

More than one monomer

sigmoidal

Question 3

P50

Answer 3

concentration of substrate that gives half saturation

Question 4

Hill plot

Answer 4

log(y/(1-y0) = nlog(p02) - nlogP50
n=gradient, 1 = noncooperative, >1 cooperative
or log(V/Vmax-v) vs log(V) (allosteric enzymes)

Question 5

R state

Answer 5

relaxed = reactive

Question 6

T state

Answer 6

tense = not reactive

Question 7

MWC model

Answer 7

concerted, symmetrical - all T or all R. One binds -> all R.
+ve homotropic effects, +ve and -ve heterotropic effects.
biased to T

Question 8

Homotropic effectors

Answer 8

binding of molecule to protein/enzyme effects binding of the same type of molecule at a different site

Question 9

Heterotropic effects

Answer 9

affects binding of a different molecule

Question 10

KNF model

Answer 10

hybrid model
TT->RT->RR
+ve and -ve homo and hetertropic effects

Question 11

ATCase

Answer 11

aspartate carbanoyltransferase
Carbanoyl phosphate + L-asp -> tetrahedral intermediate -> phosphate + N-carbamoyl-L-asp -> CTP
12 proteins - C (catalytic) or R (regulatory)

Question 12

Y

Answer 12

Saturation/fractional occupancy

= [LR]/[R] + [LR] = [L]/[L]+Kd

Question 13

ATCase-PALA complex structure

Answer 13

C1 and C4 open up and shift 12A 
10º rotation 
Active site - histidine, arg and ser/thr
Equatorial domain - rotates to bind to aspartame's, C1 and C4 loop bands break, free up catalytic site -> T state
NWC model

Question 14

protein ligand interactions

Answer 14

small and large molecules
ligands -> (proteins, carbohydrates, nucleic acids, peptides)
exchange substrates
proteins bind non specifically

Question 15

Qualitative

Answer 15

if things bind

faster, high-throughput, error prone, false +ve and -ve

Question 16

quantitative

Answer 16

how tightly something binds

more accurate, slower expensive

Question 17

EC50/IC50

Answer 17

half maximal effective concentration

mmol=weak, nmol=strong

Question 18

Differential scanning fluprimetry

Answer 18

folded protein + heat (PCR) + SYPRO orange -> unfolding, hydrophobic dyed -> find MP (deltaTm)
Tm = fluorescence maximum

Question 19

Kd

Answer 19

[R][L]/[RL] =KL =1/Ka

small=bound tightly

Question 20

Ka

Answer 20

association constant [RL]/[R][L]

Question 21

kon/koff

Answer 21

rate of R+L->RL

[R][L] kon = [RL] koff

Question 22

Differential scanning fluorometry pros/cons

Answer 22

inexpensive, fast, easy, high throughput, fails easily

semi-quantitative

Question 23

Equilibrium dialysis pros and cons

Answer 23

inexpensive, easy ligand assay required, R and L separated by membrane, lots of material
quantitative

Question 24

ITC pros and cons

Answer 24

expensive equipment, low running costs, need lots of material, accurate, low throughput
quantitative, gold standard

Question 25

SPR pros and cons

Answer 25

accurate, expensive, high running costs, tricky set up, valuable data
quantitative

Question 26

equilibrium dialysis

Answer 26

two cell chamber - receptor and assay chambers
moves across dialysis membrane from assay chamber -> eq
assay to see how much ligand remains
Kd/Ka, stoichiometry, Y

Question 27

Bmax

Answer 27

maximum bound ligand concentration

=Rtot of 1:1 stoichiometry

Question 28

Scatchard Plot

Answer 28

equilibrium dialysis
B/F = Bmax/KL - B/KL
y = b + mx

Question 29

ITC isothermal calorimetry

Answer 29

protein-small molecule interaction
measure temperature changes when ligand and sample mixed via needle injection -> heat event until saturated
Ka and Kd, stoichiometry, enthalpy, entropy, heat capacity

Question 30

SPR surface plasmon resonance

Answer 30

polarised light into prism, resonance signal changes as substrate binds.
Gives curve with association, dissociation, regeneration etc.

Question 31

Enzymes are what type of molecule

Answer 31

most are proteins apart from catalytic RNA and ribozymes

Question 32

catalysis strategies

Answer 32

geometric (proximity and orientation)
acid-base - H+
covalent
redox and radicle (metal ions)
substrate assisted
cofactors and activated groups
transition state stabilisation

Question 33

Lysozyme

Answer 33

NAM-NAG polysaccharide substrate used for cell wall remodelling (E. coli)
cleaves -o- via hydrolysis

Question 34

Lysozyme crucial concepts

Answer 34

crystal structures (mutated) used to find mechanism
half chiar configuration
18-O labelling, fluorinated substrate analogue
Sn2 attack + inversion (double displacement)

Question 35

Lysozyme residues

Answer 35

Trp63 - discriminate NAG
Glu35 - discriminate NAG, acid catalyst to bridging oxygen, then base to stabilise H2O
Val109 - NAM, stabilise half chair
Asp52 - NAM, stabilise half chair, nucleophile to carbocation, leaving group

Question 36

Weak interactions

Answer 36

need many = good - does not create energy well
specificity and reversibility
Induced fit = lover transition energy

Question 37

PET

Answer 37

Breaks down plastic to monomers for resynthesis via hydrolysis
catalytic triad, create mutations around to lower distance between triad (red trace)
Add disulphide bond for stability (cysteine)

Question 38

Enzyme improvement

Answer 38

increase substrate affinity, catalytic turnover and stability.
Essential features vs allowed variation

Question 39

catalytic triad

Answer 39

serine nucleophilic attacks at cleavage point
His deprotonates Ser
Aspartic acid H bonds to his to stabilise