Antonio Ariza

Question 1

what are the factors determining the acidity of an organic compound Y-H

Answer 1

Y-H bond strength
electronegativity of Y
nature of the solvent eg. environments hydrophobicity
Factors that stabilize Y– (conjugate base)
compared to YH (acid)

Question 2

Handleson hasselbalch equation?
what can it be used for?

Answer 2

pH = pKa + log10([A-]/[HA])
lets us compare the ratio of acid [HA] to base [A-]

Question 3

what is the difference between an acid and a base, 2 types of bases

Answer 3

acid can donate a proton/base can accept a proton either as an A- or has a lone pair

Question 4

where does ribonuclease cut

Answer 4

after a pyrimidine U or C at P-O5’ bond

Question 5

what is the intermediate of ribonuclease A

Answer 5

a 2’-3’-cyclic compound
this means that the O bound to P are also bound to the 3’ and 2’ of the ribose

Question 6

what are the catalytic active residues in ribonuclease A

Answer 6

His119, His12

Question 7

describe the structure of ribonuclease A

Answer 7

3-stranded V-shaped anti-parallel beta sheet an 3 short alpha helices crosslinked by be disulphide bridges
active site in a deep cleft

Question 8

what are the active site residues of ribonuclease A

Answer 8

His119, His12 - mechanism
Lys41 - stabilises the negatively charged phosphate intermediate (oxyanion hole)

Question 9

what are the specific pocket residues of ribonuclease A

Answer 9

Phe120 - vdW contacts with RNA base
Ser123, Thr45 - H bond

Question 10

what makes the ribonuclease A active site specific to pyrimidines

Answer 10

it is too small for purines

Question 11

what are the essential residues in chymotrypsin

Answer 11

Ser195, His57

Question 12

What kind of enzyme is chymotrypsin, where does it cleave

Answer 12

a serine protease, after large aromatic residues (Phe, Tyr, Trp)

Question 13

what is a serine protease

Answer 13

it cleaves proteins, the mechanism uses an essential serine residue

Question 14

how was the crucial AA in chymotrypsin identified

Answer 14

modification of serine with PMSF totally inhibited the enzyme
DIPF also blocks serine proteases and related molecules e.g. acetyl cholinesterase in CNS transmission

Question 15

what is the role of Asp102 in Chymotrypsin

Answer 15

it is -ve so stabilises the +ve form of His57 allowing it to grab Ser195’s proton allowing it to act as a nucleophile

Question 16

where does the protease trypsin cleave

Answer 16

after long +ve aa (Lys, Arg)

Question 17

where does the protease elastase cleave

Answer 17

after small hydrophobic AAs (Ala, Val, Thr)

Question 18

what is the inactive form of chymotrypsin

Answer 18

chymotripsinogen - full protein

Question 19

what activates chymotripsinogen to become π-chymotrypsin

Answer 19

trypsin

Question 20

what is π-chymotrypsin

Answer 20

π-chymotrypsin only cleaved between residues 15 and 16 (leaving 15-C(-O-)=O and H3N+-16)
meaning it is able to activate π-chymotrypsin to form fully active proteins

Question 21

what is the fully active form of chymotrypsin

Answer 21

α-chymotrypsin - cleaved at
13-16 - leaving 13-C(-O-)=O and H3N+-16
and 146-149 leaving 146-C(-O-)=O and H3N+-149

Question 22

what is the catalytic result of the cleavage of chymotripsinogen by trypsin

Answer 22

there is now an N-terminal NH3+ group on Ile 16 which pairs to the side chain O- on Asp194 this alters the conformation of the main chain between residues 193-195
this forms the correct geometry of the catalytic triad

and orients the main chain amides of residues 193-195 forming the oxyanion hole

Question 23

why is chymotripsinogen not active

Answer 23

neither the catalytic triad nor the oxyanion hole are fully formed

Question 24

what is an example of convergent evolution with chymotrypsin

Answer 24

Subtilisin is a bacterial protease, it has an unrelated sequence and structure but has the same catalytic triad - Asp, His, Ser

Question 25

what a threonine peptidase

Answer 25

performs catalysis with a threonine rather than a serine nucleophile an asp and lys hold the thr in place the lys acts as a base as the hydrophobic environment allows it to be deprotonated

Question 26

in Mandelate racemase which residue removes an L proton and what does it therefore act as

Answer 26

Lys166 acts as a base

Question 27

in Mandelate racemase which residue returns a D proton and what does it therefore act as

Answer 27

His 297 acts as an acid

Question 28

what is the basis of the ribonuclease A mechanism

Answer 28

E- flow from the His12 Base (N:) to the His119 Acid (NH+) through cutting the P-O5' bond to form a cyclic intermediate where POs are bound to the 3' and 2' of the ribose E- flow back from His119 Base (N:) to His12 acid (NH+) using a water molecule, replacing the 2' with an hydroxyl and and the 5' O with a hydroxyl too This reforms the catalyst

Question 29

by what kind of reaction do proteases work

Answer 29

hydrolysis (addition of water)

Question 30

naming of specificity pockets + residues

Answer 30

P1 (protein 1) goes with S1 (specificity pocket 1) S1 S2 S3 etc. upstream of scissile bond S1' S2' s3' downstream

Question 31

what is a zymogen, give example

Answer 31

inactive enzyme precursor eg. chymotripsinogen to chymotrypsin

Question 32

what chemical can be used to modify serine resulting in inhibition of serine proteases

Answer 32

PMSF - phenyl methane sulphonyl fluoride

Question 33

what is the role of the catalytic triad

Answer 33

can stabilise the charges needed for the catalytic activity eg. chymotrypsin -ve Asp stabilises +ve His to grab Ser's proton

Question 34

what is the role of the oxyanion hole

Answer 34

orientate the product to be in a specific position

Question 35

how are serine proteases specific to certain amino acids

Answer 35

the AAs in the active site can be big, small, hydrophobic, charged etc.

Question 36

what type of sidechains is chymotrypsin specific to

Answer 36

large aromatic Phe Tyr Trp all residues have to fit in the specificity pocket for it to cut

Question 37

what type of sidechains is trypsin specific to

Answer 37

+ve charged Lys Arg all residues have to fit in the specificity pocket for it to cut

Question 38

what type of sidechains is elastase specific to

Answer 38

small hydrophobic Ala Val Thr all residues have to fit in the specificity pocket for it to cut

Question 39

definition of sequence identity

Answer 39

% of AAs which are identical between the 2 proteins

Question 40

definition of sequence similarity

Answer 40

% identical + % similar (eg. polar etc.?)

Question 41

why are proteases often expressed as a zymogen? what needs to be done to make it active?

Answer 41

their effects would be unspecific (they would cut too much protein) a part needs to be cleaved or inhibitor removed disulphide bonds can keep parts of the protein together despite being cut

Question 42

which type of enzymes use a nucleophilic attack mechanism

Answer 42

threonine peptidases

Question 43

which is a stronger acid, serine or cysteine? so which has a higher pKa?

Answer 43

cysteine cysteine is -SH serine is -OH O is more electronegative than S (valence electrons closer to the nucleus) so S is worse at holding on to H serine has a higher pKa - holds on to the proton better

Question 44

what is the role of the given AA in [AA] protease

Answer 44

it is the active AA

Question 45

what is a common role of a histidine in a protease

Answer 45

abstracting a H from the active site AA to turn it into its active form (nucleophile?)

Question 46

what is the job of mandelate racemase

Answer 46

catalyses the inversion of the chiral centre of mandelate (R <--> S)