Lecture 8: Mechanisms Flashcards by B. Smith

What type of enzyme is HIV protease?

aspartyl protease

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Why is HIV protease needed?

has to cut a polyprotein in order for individual proteins to do work for HIV

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Where does HIV protease bind?

at locations with Phe and Pro

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How does HIV mechanism work?

Water is activated to attack onto the carbonyl carbon to form a tetrahedral intermediate which collapses

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How is water activated in HIV mechanism?

through base catalysis with an aspartyl residue

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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

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Drugs to treat HIV

transition state analogs

have bulky groups like Phe and Pro

resemble a N-group and Carbonyl

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Transition state analogs

resemble the transition state more than the ground state of the substrate

act as an inhibitor

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Penicillin function

blocks transpeptidase which synthesizes the peptidoglycan cell wall

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B-lactamase function

breaks down pencilin

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Structure of peptidoglycan

has peptide chain and sugar part

amino acids are in the D-conformation

transpeptidase can bind onto the peptide chain

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Transpeptidase function

binds onto peptidoglycan and allows peptides to covalently attach to each other and build the cell-wall

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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

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Augmentin

antibiotic combination

combines the use of regular penicillin with clavulanic acid

able to knock out transpeptidases and B-lactamases

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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

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What is penicillin to B-lactamase?

a substrate

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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