Chymotrypsin and Serine Proteases Lecture Review

This set of flashcards covers the lecture notes on Chymotrypsin and Serine Proteases, including their evolution, catalytic mechanisms, structural classifications, and regulation by Serpins and chemical inhibitors.

What is the primary function of serine proteases?

Serine proteases are a family of enzymes that cleave peptide bonds in proteins.

Which specific amino acid is almost always the nucleophile in the active site of serine proteases?

$$Serine$$

Serine proteases are cited as a good example of which two types of evolution?

Convergent and Divergent Evolution

Name four complex physiological cascades coordinated by serine proteases.

Digestion, immune response, blood coagulation, and reproduction

How did the Chymotrypsin-like family evolve new physiological functions from a single common ancestor?

Through gene duplication followed by minor mutations in the enzyme's binding pocket that changed its shape and charge.

What type of residues does Chymotrypsin cleave?

Large hydrophobic or aromatic residues

Which pancreatic digestive enzyme targets positively charged residues like $Lysine$ or $Arginine$?

$$Trypsin$$

What is the substrate specificity of Elastase?

Small, non-charged residues

What is the physiological role of Thrombin?

It activates fibrinogen to form blood clots.

Which protease is responsible for dissolving blood clots?

$Plasmin$ (also known as $Fibrinolysin$)

What is the function of Complement $C1$ in the immune system?

It triggers the complement cascade, leading to cell lysis.

Which protease enables sperm penetration of ova during reproduction?

$$Acrosomal\,protease$$

What are Keratinase and Collagenase responsible for?

The digestion of hair, skin, and connective tissue.

Which protease is involved in the dissolution of the cocoon during moth metamorphosis?

$$Cocoonase$$

What is the function of Acetylcholinesterase ($AcHe$)?

It hydrolyzes the neurotransmitter $acetylcholine$.

Name a non-specific protease produced by $Bacillus\,subtilis$.

$$Subtilisin$$

What are the two completely unrelated structural scaffolds that convergently evolved the $Ser-His-Asp$ catalytic triad?

Chymotrypsin-like ($beta-barrels$) and Subtilisin-like ($alpha/beta\,sheets$)

Describe the structure of Chymotrypsin-like proteases.

Two $beta-barrels$ stacked on top of each other.

In the Chymotrypsin-like scaffold, where is the nucleophilic $Serine$ located?

In the lower $beta-barrel$

Where are the $Histidine$ and $Aspartate$ residues located in the Chymotrypsin-like scaffold?

In the top $beta-barrel$

What structural fold describes the Subtilisin-like family?

An $alpha/beta$ open twisted sheet (central $beta-sheet$ surrounded by multiple $alpha-helices$)

What are the components of the catalytic triad in the Chymotrypsin family?

$Serine$, $Histidine$, and $Aspartic\,Acid\,(Asp)$

What variation of the catalytic triad does Acetylcholinesterase ($AcHe$) use?

It uses $Glutamic\,Acid\,(Glu)$ instead of $Aspartate$.

What is the role of $Histidine$ in the catalytic triad?

It acts as a base to abstract (deprotonate) a proton from the adjacent $Serine$.

How do $Aspartic\,Acid$ or $Glutamic\,Acid$ assist the catalytic triad?

They modulate the $pKa$ of the $Histidine$, making it a significantly stronger base.

The $pKa$ of the $Histidine$ imidazole nitrogen increases from approximately $7$ to what value during activation?

$$\approx 12$$

Why must $Serine$ lose its proton in the catalytic mechanism?

To be transformed into a highly reactive nucleophile for attacking the carbonyl carbon of the substrate.

Where is Chymotrypsin produced in the body?

The pancreas

Which specific bulky and aromatic amino acids does Chymotrypsin target?

$Tryptophan$, $Tyrosine$, $Phenylalanine$, $Leucine$, and $Methionine$

Chymotrypsin cleaves the peptide bond on which side of the target residue?

The $C-terminal$ (carboxyl) side

What is the name of the inactive precursor of Chymotrypsin?

$$Chymotrypsinogen$$

Which enzyme first cleaves $Chymotrypsinogen$ to begin its activation?

$$Trypsin$$

Define a zymogen.

An inactive precursor of an enzyme that requires proteolytic cleavage for activation.

Why is Chymotrypsin synthesized as a zymogen?

To prevent the enzyme from digesting the pancreas where it is produced.

What chemical reaction does Chymotrypsin facilitate to cleave peptide bonds?

Hydrolysis

Which substrate analog is used in enzyme assays to produce a yellow color upon cleavage by Chymotrypsin?

$N-acetyl-L-phenylalanine\,p-nitrophenyl\,amide$ (or ester)

What determines the specificity of a serine protease for its substrate?

The binding (specificity) pocket

In Trypsin, what residue is found at the bottom of the specificity pocket to attract positive charges?

$$Asparate\,(D102)$$

What is the first step of the six-step reaction mechanism of Chymotrypsin?

Substrate attaches to the enzyme at the main chain binding site and specificity pocket ($ES\,formation$).

What is formed immediately after the nucleophilic attack of $S195$ on the carbonyl carbon?

A tetrahedral intermediate ($transition\,state$)

How is the negative charge on the tetrahedral intermediate stabilized in Chymotrypsin?

By the $oxyanion\,hole$

Which residue is specifically numbered $S195$ in Chymotrypsin?

The nucleophilic $Serine$

Which residue is specifically numbered $H57$ in Chymotrypsin?

The $Histidine$ of the catalytic triad

What is the name of the stable intermediate formed in step 3 where the peptide is covalently linked to the enzyme?

$$Acyl-enzyme\,intermediate$$

In the second half of the Chymotrypsin reaction, what molecule acts as the second nucleophile?

$$Water\,(H_2O)$$

What is the final step ($Step\,6$) of the Chymotrypsin mechanism?

Peptide is released while $H57$ donates a proton back to $S195$, returning the enzyme to its original state ($E+P$).

What are the two phases of Chymotrypsin kinetics observed in inhibition assays?

An initial 'burst' phase followed by a steady-state phase

What is another name for the 'burst' and 'steady-state' kinetic mechanism of Chymotrypsin?

The 'ping-pong' mechanism

At what wavelength is the light absorbance of $p-nitrophenolate$ measured?

$$405\,nm$$

What are SERPINS?

Serine Protease Inhibitors

Why must the body produce Serpins?

To tightly regulate powerful and dangerous cleavage reactions in cascades like coagulation and immune responses.

What is the 'bait' presented by a Serpin called?

The $Reactive\,Center\,Loop\,(RCL)$

How does a Serpin trap a protease?

It creates a covalent $acyl-enzyme$ intermediate and then undergoes a conformational change that physically deforms the protease.

Describe the 'S to R' transition in Serpins.

The transition from a high-energy 'stressed' state to a stable 'relaxed' state.

What structural change occurs to the Serpin’s central beta-sheet during inhibition?

The sheet increases from $4$ strands to $5$ strands as it incorporates the cleaved $RCL$.

What is the physical consequence of the S to R transition on the protease's active site?

The catalytic $Serine$ is dragged away from the $Histidine$, breaking the catalytic triad.

Which Serpin inhibits Thrombin to prevent continuous blood clotting?

$$Anti-thrombin$$

What molecule must bind to $Anti-thrombin$ to activate it?

$$Heparin$$

How does $Heparin$ activate $Anti-thrombin$?

It causes an allosteric change that exposes the arginine 'bait' on the $RCL$, which was previously hidden.

What is the function of the Serpin $Anti-trypsin$?

It regulates inflammation.

Which Serpin regulates the cleavage of $C2$ and $C4$ in the complement system?

$$C1-inhibitor$$

What is unique about the regulation of $Plasminogen\,activator\,inhibitor-1\,(PAI-1)$?

It has a built-in timer; it spontaneously transitions to a latent relaxed state over time ($auto-inactivation$).

What molecule stabilizes $PAI-1$ in its active, stressed state?

$$Vitronectin$$

What is a 'catalytic trap' in the context of serine protease homologs?

A mutated enzyme that has lost its active site but retains its binding pocket, allowing it to act as a regulatory protein.

What was the original evolutionary role of $Protein\,Z$?

It was a serine protease involved in coagulation.

What is the current function of $Protein\,Z$?

It acts as a co-factor that helps $PZI$ ($Factor\,Xa\,inhibitor$) bind to its target.

Describe the function of the snake venom protein $VaaSPH-1$.

It is a serine protease homolog that lost its active site and now functions as a potent anti-coagulant.

What is the main advantage of gene duplication for enzyme evolution?

It provides a redundant gene copy that can accumulate mutations and develop new target specificities without harming the organism.

How does $TEV\,protease$ differ from standard serine proteases?

It evolved divergently to use $Cysteine$ as the nucleophile instead of $Serine$.

Identify a convergently evolved cysteine protease mentioned in the notes.

$$Papain$$

Why does $Papain$ lack an $Aspartate$ in its active site?

$Cysteine$ is a stronger acid than $Serine$, so $Histidine$ can extract its proton without the help of $Aspartate$.

What is the medical use of $Heparin$?

It is used as an anti-coagulant drug to treat blood clotting diseases.

What is the cause of certain 'Serpin diseases'?

Misfolding and polymerization, where the $RCL$ of one Serpin inserts into the beta-sheet of another.

What are some examples of chemical organophosphorus nerve agents?

$Sarin$, $VX$, and $Novichok$

What do nerve agents like $Novichok$ mimic to fool the enzyme?

A peptide bond

How do nerve agents permanently inactivate serine proteases?

They form a highly stable covalent complex with the catalytic $Serine$ that cannot be hydrolyzed.

Where is the artificial bond of a nerve agent stabilized within the enzyme?

The $oxyanion\,hole$

Nerve agents like $Novichok$ target which two systems simultaneously?

The immune ($complement$) and blood clotting systems.

What are the three components of a 'Plug and Play' catalytic triad?

Nucleophile, base, and acid.

Which enzymes cleave different bonds in beta-lactams using triad mechanisms?

$Penicillin\,acylase$ and $Beta-lactamase$

The Chymotrypsin-like family is a prime example of which evolutionary process?

Divergent evolution

In the Chymotrypsin mechanism, what happens when the first tetrahedral intermediate collapses?

$H57$ donates a proton to the nitrogen of the leaving peptide fragment, and the $C-terminal$ fragment diffuses away.

Why is the $OH^-$ from water a poorer leaving group than the $C-terminal$ fragment?

This ensures that when the second tetrahedral intermediate collapses, the $Serine$ side chain is the one that leaves.

Upon binding of the target protein, which specific residue in Trypsin forms a low-barrier hydrogen bond?

$$Asparate\,102\,(D102)$$

What is the effect of the low-barrier hydrogen bond on $Histidine\,57\,(H57)$?

It increases the $pKa$ of its imidazole nitrogen, making it a powerful general base.

In $Subtilisin$, which residue numbers make up the catalytic triad?

$Serine\,221$, $Histidine$, and $Aspartate$ (Notes specify $Ser221$ and $Asn155$ in the hole).

What role do $NH$ groups play in the $oxyanion\,hole$ of $Subtilisin$?

They stabilize the negative charge that develops on the peptide bond attacked by the nucleophile.

What happens to the activity of a protease if its $Serine$ and $Histidine$ residues are lost?

It loses enzymatic activity but can gain regulatory functions (becoming a catalytic trap).

What is the structural commonality between all enzymes in the Chymotrypsin-like family?

They all share the same structural scaffold of two $beta-barrels$ stacked on top of each other.

Which Serpin regulates tissue remodeling?

$$Plasminogen\,activator\,inhibitor-1$$

The $C1-inhibitor$ prevents the continuous cleavage of which two complement components?

$C2$ and $C4$

In the inactive state of $Anti-thrombin$, where is the Arginine residue?

It is hidden and facing away on the $Reactive\,Center\,Loop$.

What happens to the functional supply of an inhibitor when categorization misfolding occurs?

The functional supply is destroyed as Serpins link together into polymer chains.

The 'ping-pong' mechanism of Chymotrypsin consists of which two chemical stages?

Acylation and deacylation

What are the subunits of the $C1$ complex that contain active serine proteases?

$C1r$ and $C1s$

What is the term for the bond that is intended to be cleaved by the protease?

The $scissile\,bond$

Where does the water molecule place itself in the Chymotrypsin mechanism ($Step\,4$)?

Next to $H57$ and the $acyl-enzyme\,intermediate$ at $S195$.

Which amino acid's side chain folds back onto the main chain, making it 'uncleavable' in certain contexts?

$$Proline$$

What is the metabolic fate of the Serpin-protease inactive complex?

It is sent to the liver to be broken down and destroyed.

Which specific residue acts as the 'acid' in the Chymotrypsin catalytic triad?

$$Asparate\,102\,(D102)$$