Lecture 8: Mechanisms Flashcards
What type of enzyme is HIV protease?
aspartyl protease
Why is HIV protease needed?
has to cut a polyprotein in order for individual proteins to do work for HIV
Where does HIV protease bind?
at locations with Phe and Pro
How does HIV mechanism work?
Water is activated to attack onto the carbonyl carbon to form a tetrahedral intermediate which collapses
How is water activated in HIV mechanism?
through base catalysis with an aspartyl residue
What are the aspartyl residues used for in HIV protease?
one is used for base catalysis
the other is used to stabilize the negative oxygen in the tetrahedral intermediate
Drugs to treat HIV
transition state analogs
have bulky groups like Phe and Pro
resemble a N-group and Carbonyl
Transition state analogs
resemble the transition state more than the ground state of the substrate
act as an inhibitor
Penicillin function
blocks transpeptidase which synthesizes the peptidoglycan cell wall
B-lactamase function
breaks down pencilin
Structure of peptidoglycan
has peptide chain and sugar part
amino acids are in the D-conformation
transpeptidase can bind onto the peptide chain
Transpeptidase function
binds onto peptidoglycan and allows peptides to covalently attach to each other and build the cell-wall
Structure of penicillin
4 membered ring (B-lactam ring) fused to a 5 membered ring (thiazolidine ring)
Structure mimics the natural substrate of transpeptidase
Rings look like a dipeptide with alternating N-groups and carbonyl groups
Augmentin
antibiotic combination
combines the use of regular penicillin with clavulanic acid
able to knock out transpeptidases and B-lactamases
Clavulanic acid
looks like penicillin and interacts with B-lactamase
however, clavulanic acid rearranges to form a structure that cannot be detached from B-lactamase
What is penicillin to B-lactamase?
a substrate
What is penicillin to transpeptidase?
an inhibitor
Difference between B-lactamase and transpeptidase mechanism?
When B-lactamase forms covalent bond with penicillin it can detach
Water is allowed into B-lactamase’s active site which disbonds penicillin
What type of enzyme is chymotrypsin?
a serine protease that helps break down proteins in foods
Where does chymotrypsin cut?
after aromatic amino acids
Trp, Phe, Tyr
What do you except the active site of chymotrypsin to be?
large and hydrophobic in order to allow aromatic rings into the active site
Characteristics of chymotrypsin
in inactive form is a zymogen, chymotrypsinogen
has disulfide bonds
possesses a catalytic triad
pH profile of chymotrypsin
activity is maximized at pH=8
Km is lowest (greater affinity) below 8
Kcat is fastest above 8
How do you determine Kcat/Km?
use a low substrate concentration
What happens to chymotrypsin at high pHs?
the salt bridge between Ile16 and Asp194 is disrupted
amino groups are deprotonated and the salt bridge goes away
this causes the active site to close
What happens to chymotrypsin at low pHs?
His 57 is protonated and cannot interact in catalysis
Overall mechanism of chymotrypsin
1) Acylation: peptide bond is cleaved and an ester linkage is formed between the protein and enzyme
2) Deacylation: ester linkage is hydrolyzed, enzyme is regenerated
Catalytic triad of chymotrypsin
Ser, His, and Asp
What does serine do in chymotrypsin intially?
acts as a nucleophile after the hydrogen is removed from the -OH group
What does histidine do in chymotrypsin intially?
acts as a general base to pull off serine’s hydrogen
What does aspartate do in chymotrypsin initially?
stabilizes the formation of a positive charge on histidine
makes it more likely for histidine to pull off a hydrogen
raises the pKa of histidine as histidine is more likely to have a proton
What are the serines on either side of the active site used for?
as one performs a nucleophilic attack, the serine directly on the other side stabilizes the tetrahedral intermediate that forms
Oxyanion hole
made up of serine and glycine to stabilize the tetrahedral intermediate in chymotrypsin
Acylation step
serine attacks the carbonyl and forms a tetrahedral intermediate that is stabilized by the oxyanion hole
the tetrahedral collapses and the first product breaks off
the first product’s N-group is stabilized by the H+ on histidine (general acid catalysis)
left with the serine attached to the other part of the substrate
Deacylation step
water comes into the active site and histidine acts as a general base to activate water
water attacks the carbonyl and forms second tetrahedral intermediate (stabilized by oxyanion hole)
positively charged histidine donates a proton back onto serine (general acid)
second tetrahedral product collapses and releases final product
List all the intermediates of the chymotrypsin mechanism
1) Tetrahedral number 1
2) Acyl-enzyme
3) Tetrahedral number 2
Are the transition states the intermediates of chymotrypsin mechanism?
No! Transition states are just when energy is the highest
Do not neccessarily line up with intermediates
How many steps does HIV-protease have?
one step
What is substrate of transpitadase?
the D-Ala D-Ala section of the peptide chain in peptidoglycan
How many steps does transpeptidase have?
2 steps
first have an acylation step
then, have a deacylation step but with another amino acid (rather than water in chymotrypsin mechanism)
What type of inhibitor is penicillin?
suicide inhibitor
forms a permanent covalent bond with transpeptidase
What is B-lactamase mechanism very similar to?
chymotrypsin mechanism
almost the same two steps
