Antonio Ariza Flashcards
what are the factors determining the acidity of an organic compound Y-H
Y-H bond strength
electronegativity of Y
nature of the solvent eg. environments hydrophobicity
Factors that stabilize Y– (conjugate base)
compared to YH (acid)
Handleson hasselbalch equation?
what can it be used for?
pH = pKa + log10([A-]/[HA])
lets us compare the ratio of acid [HA] to base [A-]
what is the difference between an acid and a base, 2 types of bases
acid can donate a proton/base can accept a proton either as an A- or has a lone pair
where does ribonuclease cut
after a pyrimidine U or C at P-O5’ bond
what is the intermediate of ribonuclease 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
what are the catalytic active residues in ribonuclease A
His119, His12
describe the structure of ribonuclease 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
what are the active site residues of ribonuclease A
His119, His12 - mechanism
Lys41 - stabilises the negatively charged phosphate intermediate (oxyanion hole)
what are the specific pocket residues of ribonuclease A
Phe120 - vdW contacts with RNA base
Ser123, Thr45 - H bond
what makes the ribonuclease A active site specific to pyrimidines
it is too small for purines
what are the essential residues in chymotrypsin
Ser195, His57
What kind of enzyme is chymotrypsin, where does it cleave
a serine protease, after large aromatic residues (Phe, Tyr, Trp)
what is a serine protease
it cleaves proteins, the mechanism uses an essential serine residue
how was the crucial AA in chymotrypsin identified
modification of serine with PMSF totally inhibited the enzyme
DIPF also blocks serine proteases and related molecules e.g. acetyl cholinesterase in CNS transmission
what is the role of Asp102 in Chymotrypsin
it is -ve so stabilises the +ve form of His57 allowing it to grab Ser195’s proton allowing it to act as a nucleophile
where does the protease trypsin cleave
after long +ve aa (Lys, Arg)
where does the protease elastase cleave
after small hydrophobic AAs (Ala, Val, Thr)
what is the inactive form of chymotrypsin
chymotripsinogen - full protein
what activates chymotripsinogen to become π-chymotrypsin
trypsin
what is π-chymotrypsin
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
what is the fully active form of chymotrypsin
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
what is the catalytic result of the cleavage of chymotripsinogen by trypsin
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
why is chymotripsinogen not active
neither the catalytic triad nor the oxyanion hole are fully formed
what is an example of convergent evolution with chymotrypsin
Subtilisin is a bacterial protease, it has an unrelated sequence and structure but has the same catalytic triad - Asp, His, Ser
what a threonine peptidase
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
in Mandelate racemase which residue removes an L proton
and what does it therefore act as
Lys166
acts as a base
in Mandelate racemase which residue returns a D proton
and what does it therefore act as
His 297
acts as an acid
what is the basis of the ribonuclease A mechanism
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
by what kind of reaction do proteases work
hydrolysis (addition of water)
naming of specificity pockets + residues
P1 (protein 1) goes with S1 (specificity pocket 1)
S1 S2 S3 etc. upstream of scissile bond
S1’ S2’ s3’ downstream
what is a zymogen, give example
inactive enzyme precursor
eg. chymotripsinogen to chymotrypsin
what chemical can be used to modify serine resulting in inhibition of serine proteases
PMSF - phenyl methane sulphonyl fluoride
what is the role of the catalytic triad
can stabilise the charges needed for the catalytic activity
eg. chymotrypsin
-ve Asp stabilises +ve His to grab Ser’s proton
what is the role of the oxyanion hole
orientate the product to be in a specific position
how are serine proteases specific to certain amino acids
the AAs in the active site can be big, small, hydrophobic, charged etc.
what type of sidechains is chymotrypsin specific to
large aromatic
Phe Tyr Trp
all residues have to fit in the specificity pocket for it to cut
what type of sidechains is trypsin specific to
+ve charged
Lys Arg
all residues have to fit in the specificity pocket for it to cut
what type of sidechains is elastase specific to
small hydrophobic
Ala Val Thr
all residues have to fit in the specificity pocket for it to cut
definition of sequence identity
% of AAs which are identical between the 2 proteins
definition of sequence similarity
% identical + % similar (eg. polar etc.?)
why are proteases often expressed as a zymogen? what needs to be done to make it active?
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
which type of enzymes use a nucleophilic attack mechanism
threonine peptidases
which is a stronger acid, serine or cysteine?
so which has a higher pKa?
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
what is the role of the given AA in [AA] protease
it is the active AA
what is a common role of a histidine in a protease
abstracting a H from the active site AA to turn it into its active form (nucleophile?)
what is the job of mandelate racemase
catalyses the inversion of the chiral centre of mandelate (R <–> S)
