Enzyme function Flashcards
Control of enzyme activity
gene expression, protein degradation, covalent modification (phosphorylation), allosteric control, protein binding, zymogens
Cooperativity
More than one monomer
sigmoidal
P50
concentration of substrate that gives half saturation
Hill plot
log(y/(1-y0) = nlog(p02) - nlogP50 n=gradient, 1 = noncooperative, >1 cooperative or log(V/Vmax-v) vs log(V) (allosteric enzymes)
R state
relaxed = reactive
T state
tense = not reactive
MWC model
concerted, symmetrical - all T or all R. One binds -> all R.
+ve homotropic effects, +ve and -ve heterotropic effects.
biased to T
Homotropic effectors
binding of molecule to protein/enzyme effects binding of the same type of molecule at a different site
Heterotropic effects
affects binding of a different molecule
KNF model
hybrid model
TT->RT->RR
+ve and -ve homo and hetertropic effects
ATCase
aspartate carbanoyltransferase
Carbanoyl phosphate + L-asp -> tetrahedral intermediate -> phosphate + N-carbamoyl-L-asp -> CTP
12 proteins - C (catalytic) or R (regulatory)
Y
Saturation/fractional occupancy
= [LR]/[R] + [LR] = [L]/[L]+Kd
ATCase-PALA complex structure
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
protein ligand interactions
small and large molecules
ligands -> (proteins, carbohydrates, nucleic acids, peptides)
exchange substrates
proteins bind non specifically
Qualitative
if things bind
faster, high-throughput, error prone, false +ve and -ve
quantitative
how tightly something binds
more accurate, slower expensive
EC50/IC50
half maximal effective concentration
mmol=weak, nmol=strong
Differential scanning fluprimetry
folded protein + heat (PCR) + SYPRO orange -> unfolding, hydrophobic dyed -> find MP (deltaTm)
Tm = fluorescence maximum
Kd
[R][L]/[RL] =KL =1/Ka
small=bound tightly
Ka
association constant [RL]/[R][L]
kon/koff
rate of R+L->RL
[R][L] kon = [RL] koff
Differential scanning fluorometry pros/cons
inexpensive, fast, easy, high throughput, fails easily
semi-quantitative
Equilibrium dialysis pros and cons
inexpensive, easy ligand assay required, R and L separated by membrane, lots of material
quantitative
ITC pros and cons
expensive equipment, low running costs, need lots of material, accurate, low throughput
quantitative, gold standard
SPR pros and cons
accurate, expensive, high running costs, tricky set up, valuable data
quantitative
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
Bmax
maximum bound ligand concentration
=Rtot of 1:1 stoichiometry
Scatchard Plot
equilibrium dialysis
B/F = Bmax/KL - B/KL
y = b + mx
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
SPR surface plasmon resonance
polarised light into prism, resonance signal changes as substrate binds.
Gives curve with association, dissociation, regeneration etc.
Enzymes are what type of molecule
most are proteins apart from catalytic RNA and ribozymes
catalysis strategies
geometric (proximity and orientation) acid-base - H+ covalent redox and radicle (metal ions) substrate assisted cofactors and activated groups transition state stabilisation
Lysozyme
NAM-NAG polysaccharide substrate used for cell wall remodelling (E. coli)
cleaves -o- via hydrolysis
Lysozyme crucial concepts
crystal structures (mutated) used to find mechanism
half chiar configuration
18-O labelling, fluorinated substrate analogue
Sn2 attack + inversion (double displacement)
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
Weak interactions
need many = good - does not create energy well
specificity and reversibility
Induced fit = lover transition energy
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)
Enzyme improvement
increase substrate affinity, catalytic turnover and stability.
Essential features vs allowed variation
catalytic triad
serine nucleophilic attacks at cleavage point
His deprotonates Ser
Aspartic acid H bonds to his to stabilise
