Protein Biochemistry and Enzymology

Question 1

beta-galactosidase

Answer 1

hydrolyses lactose
can also hydrolyse other galactosides

Question 2

michaelis-menten equation

Answer 2

v=vmax [S}/Km +[S]
v = velocity
vmax = maximal velocity
Km = michaelis constant - this is the substrate concentration that gives v = vmax/2

Question 3

what is an overall enzyme catalysed reaction described as?

Answer 3

Km = (k2 +kcat)/k1
Km is approximate to the affinity of k2/k1

Question 4

rate of formation of P is proportional to…
so at saturation what equation can you use?

Answer 4

v = kcat[ES]

vmax = kcat[Etot]

Question 5

what is Vmax directly proportional to?

Answer 5

[E]

Question 6

what are the two constants that describe its kinetic properties?

Answer 6

m-m eq = v = vmax [S]/Km + [S]

vmax = kcat[Etot]

kcat describes how fast the enzyme works, so the turnover

Km describes its substrate concentration dependence, approximates to affinity

Question 7

what is the specificity constant?

Answer 7

specificity constant = kcat/Km

this is the best single measure of enzzyme catalytic efficiency

Question 8

what constants would you expect for a good enzyme?

Answer 8

kcat - high
Km - low
kcat/Km - high

Question 9

lineweaver-burke plot

Answer 9

1/v = Km/Vmax 1/[S] + 1/Vmax

use like its y=mx+c

also known as a double reciprocal plot

Question 10

what are the benefits to enzyme inhibition?

Answer 10

understanding how enzymes work
producing drugs
useful for diagnosis and treatment of disease such as myasthenia gravis (progressive muscular weakness with the problem being at the neuromuscular junction -ACh)

Question 11

how can we recognise competitive inhibition?

Answer 11

the Vmax stays the same
the substrate concentration required to produce Vmax/2 is increase
the apparent value of Km increases

Question 12

what is the equation for Km apparent?

Answer 12

Km App = Km(1+[I]/Ki

Question 13

how is Ki determined?

Answer 13

[I]/([Km, apparent] - Km) = Ki

Question 14

what kind of modification can cause irreversible inhibition?

Answer 14

covalent modification

Question 15

edrophonium

Answer 15

competitive inhibitor of acetylcholinesterase
used to diagnose myasthenia gravis
effects do not last long

Question 16

what are the functions of proteins?

Answer 16

defence - hair keratin, immunity antibodies
structure - mechanical support such as silk, coordinated movement muscles
catalysis - enzymes
transport - transport of oxygen

Question 17

zwitterion

Answer 17

molecule with both positive and negative charges on the functional groups but an overall charge of 0

Question 18

amino acids

Answer 18

D-AA exist but not in proteins encoded by DNA
AA in proteins all have the L-configuration

Question 19

in hydrogen bonding, which is the better donor and which is the acceptor

Answer 19

nitrogen is a H bond donor
oxygen is a H bond acceptor

Question 20

when is a hydrogen bond strongest?

Answer 20

when linear

Question 21

properties of alpha helix

Answer 21

right handed helix
3.6 residues/turn
good H bonding

Question 22

what are helix former examples? (AAs)

Answer 22

glutamate
glutamine
alanine
histidine
methionine
leucine
lysine
arginine

Question 23

what is a helix destabiliser example? (AAs)

Answer 23

tyrosine

Question 24

what is a helix breaker example? (AAs)

Answer 24

proline - remember: the bond cannot rotate

Question 25

in what formation does silk run in?

Answer 25

antiparallel beta sheets

Question 26

silk

Answer 26

firoin has long stretches with sequence (GlyAla)n antiparallel beta sheet silk will not stretch in a longitudinal direction but is flexible laterally note: you can get parallel beta sheets

Question 27

name some AA hydrophobic side chains

Answer 27

valine leucine phenylalanine

Question 28

name some AA hydrophilic side chains

Answer 28

aspartate lysine serine

Question 29

disulphide bonds

Answer 29

covalent bonds between two cysteine side-chains

Question 30

example of ion pair bond (salt bridge)

Answer 30

aspartate and lysine

Question 31

equation for ionisation or acid base equilibria

Answer 31

K = [COO-][H+]/[COOH]

Question 32

pKa is defined as

Answer 32

pKa= -logKa pH=pKa +log10([base]/[acid]

Question 33

domain

Answer 33

globular unit formed from part of polypeptide (often have a particular function)

Question 34

HIV proteinase for poly-protein processing, what is this useful for?

Answer 34

maturation assembly

Question 35

what is the function of folding

Answer 35

structure and activity necessary for drug design necessary for protein engineering

Question 36

SDS PAGE

Answer 36

sodium dodecyl sulphate: a detergent polyacrylamide gel electrophoresis SDS unfolds the protein by coating unfolded chain stained with Coomassie Blue or silver nitrate

Question 37

purification of proteins, name the methods of separation

Answer 37

solubility - polar residues, differ due to surfaces precipitation by ammonium sulphate (salting out) - depends on polarity of the protein surface, determines how soluble in water, dissolved salt needs to be solvated by adding ammonium sulphate decreases the water available to solvate the protein, centrifuge, assess supernatant precipitate isoelectric focusing - mixture of proteins, either positive or negative, moves along pH gradient and stop at isoelectric point, stained with Coomassie Blue or silver nitrate

Question 38

what wavelength do proteins absorb UV light?

Answer 38

280nm this is due to aromatic residues

Question 39

size exclusion chromatography (SEC) (gel filtration) and separation according to size (molecular sieving)

Answer 39

SEC - cross-linked polydextran, large molecules cannot enter

Question 40

what does an SDS PAGE give, and what does an SEC give?

Answer 40

SDS PAGE - monomer size SEC - native size combination of the two indicated quaternary size

Question 41

ion exchange chromatography

Answer 41

depends on ionisable groups in protein (pH crucial) interaction of charges groups on protein with charged matrix in column polymer substituted with charged groups

Question 42

affinity chromatography

Answer 42

proteins can have a selective affinity for a particular structure