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Biochemistry II > Antonio Ariza > Flashcards

Antonio Ariza Flashcards

1
Q

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

A

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

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2
Q

Handleson hasselbalch equation?
what can it be used for?

A

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

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3
Q

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

A

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

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4
Q

where does ribonuclease cut

A

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

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5
Q

what is the intermediate of ribonuclease A

A

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

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6
Q

what are the catalytic active residues in ribonuclease A

A

His119, His12

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7
Q

describe the structure of ribonuclease A

A

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

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8
Q

what are the active site residues of ribonuclease A

A

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

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9
Q

what are the specific pocket residues of ribonuclease A

A

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

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10
Q

what makes the ribonuclease A active site specific to pyrimidines

A

it is too small for purines

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11
Q

what are the essential residues in chymotrypsin

A

Ser195, His57

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12
Q

What kind of enzyme is chymotrypsin, where does it cleave

A

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

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13
Q

what is a serine protease

A

it cleaves proteins, the mechanism uses an essential serine residue

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14
Q

how was the crucial AA in chymotrypsin identified

A

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

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15
Q

what is the role of Asp102 in Chymotrypsin

A

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

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16
Q

where does the protease trypsin cleave

A

after long +ve aa (Lys, Arg)

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17
Q

where does the protease elastase cleave

A

after small hydrophobic AAs (Ala, Val, Thr)

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18
Q

what is the inactive form of chymotrypsin

A

chymotripsinogen - full protein

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19
Q

what activates chymotripsinogen to become π-chymotrypsin

A

trypsin

20
Q

what is π-chymotrypsin

A

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

21
Q

what is the fully active form of chymotrypsin

A

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

22
Q

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

A

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

23
Q

why is chymotripsinogen not active

A

neither the catalytic triad nor the oxyanion hole are fully formed

24
Q

what is an example of convergent evolution with chymotrypsin

A

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

25
Q

what a threonine peptidase

A

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

26
Q

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

A

Lys166
acts as a base

27
Q

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

A

His 297
acts as an acid

28
Q

what is the basis of the ribonuclease A mechanism

A

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

29
Q

by what kind of reaction do proteases work

A

hydrolysis (addition of water)

30
Q

naming of specificity pockets + residues

A

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

31
Q

what is a zymogen, give example

A

inactive enzyme precursor
eg. chymotripsinogen to chymotrypsin

32
Q

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

A

PMSF - phenyl methane sulphonyl fluoride

33
Q

what is the role of the catalytic triad

A

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

34
Q

what is the role of the oxyanion hole

A

orientate the product to be in a specific position

35
Q

how are serine proteases specific to certain amino acids

A

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

36
Q

what type of sidechains is chymotrypsin specific to

A

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

37
Q

what type of sidechains is trypsin specific to

A

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

38
Q

what type of sidechains is elastase specific to

A

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

39
Q

definition of sequence identity

A

% of AAs which are identical between the 2 proteins

40
Q

definition of sequence similarity

A

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

41
Q

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

A

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

42
Q

which type of enzymes use a nucleophilic attack mechanism

A

threonine peptidases

43
Q

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

A

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

44
Q

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

A

it is the active AA

45
Q

what is a common role of a histidine in a protease

A

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

46
Q

what is the job of mandelate racemase

A

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

Biochemistry II (8 decks)

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