Enzyme function Flashcards

1
Q

Control of enzyme activity

A

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

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2
Q

Cooperativity

A

More than one monomer

sigmoidal

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3
Q

P50

A

concentration of substrate that gives half saturation

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4
Q

Hill plot

A
log(y/(1-y0) = nlog(p02) - nlogP50
n=gradient, 1 = noncooperative, >1 cooperative
or log(V/Vmax-v) vs log(V) (allosteric enzymes)
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5
Q

R state

A

relaxed = reactive

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6
Q

T state

A

tense = not reactive

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7
Q

MWC model

A

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

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8
Q

Homotropic effectors

A

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

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9
Q

Heterotropic effects

A

affects binding of a different molecule

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10
Q

KNF model

A

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

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11
Q

ATCase

A

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

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12
Q

Y

A

Saturation/fractional occupancy

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

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13
Q

ATCase-PALA complex structure

A
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
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14
Q

protein ligand interactions

A

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

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15
Q

Qualitative

A

if things bind

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

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16
Q

quantitative

A

how tightly something binds

more accurate, slower expensive

17
Q

EC50/IC50

A

half maximal effective concentration

mmol=weak, nmol=strong

18
Q

Differential scanning fluprimetry

A

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

19
Q

Kd

A

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

small=bound tightly

20
Q

Ka

A

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

21
Q

kon/koff

A

rate of R+L->RL

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

22
Q

Differential scanning fluorometry pros/cons

A

inexpensive, fast, easy, high throughput, fails easily

semi-quantitative

23
Q

Equilibrium dialysis pros and cons

A

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

24
Q

ITC pros and cons

A

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

25
SPR pros and cons
accurate, expensive, high running costs, tricky set up, valuable data quantitative
26
equilibrium dialysis
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
27
Bmax
maximum bound ligand concentration | =Rtot of 1:1 stoichiometry
28
Scatchard Plot
equilibrium dialysis B/F = Bmax/KL - B/KL y = b + mx
29
ITC isothermal calorimetry
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
30
SPR surface plasmon resonance
polarised light into prism, resonance signal changes as substrate binds. Gives curve with association, dissociation, regeneration etc.
31
Enzymes are what type of molecule
most are proteins apart from catalytic RNA and ribozymes
32
catalysis strategies
``` geometric (proximity and orientation) acid-base - H+ covalent redox and radicle (metal ions) substrate assisted cofactors and activated groups transition state stabilisation ```
33
Lysozyme
NAM-NAG polysaccharide substrate used for cell wall remodelling (E. coli) cleaves -o- via hydrolysis
34
Lysozyme crucial concepts
crystal structures (mutated) used to find mechanism half chiar configuration 18-O labelling, fluorinated substrate analogue Sn2 attack + inversion (double displacement)
35
Lysozyme residues
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
36
Weak interactions
need many = good - does not create energy well specificity and reversibility Induced fit = lover transition energy
37
PET
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)
38
Enzyme improvement
increase substrate affinity, catalytic turnover and stability. Essential features vs allowed variation
39
catalytic triad
serine nucleophilic attacks at cleavage point His deprotonates Ser Aspartic acid H bonds to his to stabilise