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113 Cards in this Set

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  • Back
What are the four classes of enzymes?
1. serine proteases
2. carbonic anhydrases
3. restriction endonucleases
4. nucleoside monophosphate (NMP) kinases
Which class(es) of enzymes require the addition of water to a substrate?
1. serine proteases
2. carbonic anhydrases
3. restriction endonucleases
Which class(es) of enzymes do require the prevention of water to a substrate?
4. nucleoside monophosphate (NMP) kinase
What is an example of a serine protease?
chymotrypsin
What is the "challange" of a serine protease (such as chymotrypsin)?
promote a reaction that is almost immeasurably slow at neutral pH in the absence of a catalyst.
What is the "challange" of carbonic anhydrase?
To achieve a high absolute rate of reaction
What is the "challange" of restriction endonucleases?
Attaining a high degree of specificity
What is the "challange" of NMP kinases?
To transfer a phosphoryl group from ATP to a nucleotide and not to water.
What are the four common strategies to catalyze specific reactions?
1. covalent catalysis
2. general acid-base catalysis
3. catalysis by approximation
4. metal ion catalysis
What is covalent catalysis?
The active site contains a reactive groupe (usually powerful nucleophile) that becomes temporarily covalently attached to a part of the substrate during catalysis.
How does general acid-base catalysis work?
A molecule other than water plays the role of proton donor or acceptor.
Chymotrypsin: uses His as a base to enhance teh nucleophilic power of serine
Carbonic anhydrase: His facilitates removal of H+ from zinc-bound water to generate OH molecule.
What is catalysis by approximation?
Includes two distinct substrates.
NMP kinase: brings two nucleotides together to facilitate the transfer of a phosphoryl group from one nucleotide to another
Who dow metal ion catalysis work?
Metal ions can:
- facilitate the formation of nucleophiles by direct coordination
- may serve as electrophile - stabilizes neg charge on reaction intermediate
- may serve as bridge b/w enzyme and substrate: holds substrate into conformation appropriate for catalysis.
What do proteases do?
They cleave proteins hydrolysis: addition of a molecule of water to a peptide bond.
Why do we need proteases?
Hydrolysis of peptide bonds are thermodynamically favored, but extremely slow. Proteases speed up the reaction from 100 years to milliseconds.
What is responsible for the kinetic stability of peptide bonds?
Resonance structure: gives planarity and structural stability (resistance to hydrolysis)
What stregthens the carbon-nitrogen bond in a protein?
Double bond character (resonance).Carbon is normally unreactive - needs an enzyme to facilitate nucleophilic attack of carbonyl group.
Where does chymotrypsin cleave peptide bonds?
Selectively on the carboxyl side of large hydrophobic amino acids:
- trp
- tyr
- phe
- met
What type of catalysis does chymotrypsin utilize?
Covalent catalysis: nucleophilic attack on unreactive carbonyl carbon of the substrate. This nucleophile become covalently attached to the substrate briefly in the course of catalysis.
What residue is extremely reactive in chymotrypsin?
Serine residue (one of the twenty-eight) which lies in the active site.
Does chymotrypsin obey M-M kinetics?
Under steady-state, yes.
What can proteases hydrolyze?
Mostly amides, also esters (usually in vitro)
What method can be used to moniter enzyme-substrate interaction?
Stop-flow method
What are the steps involved in chymotrypsin hydrolysis of peptide linkages?
1. acyl group of the substrate becomes covalently attached to the enzyme; enzyme-acetyl complex is released = acyl-enzme intermediate

2. acyl-enzyme intermediate is hydrolyzed to release the carboxylic acid component of the substrate and regenerate the free enzyme.
How can we see the two stages of cleavage by chymotrypsin?
Stopped-flow method: shows a burst phase and a stady-state phase. This is viewed on a graph as seconds after mixing vs. absorbance: steep slope and then much flatter.
What is chymotrypsinogen?
The name of the single polypeptide of chymotrypsin: chymotrypsin=3 of these linked by disulfide bonds
List the 8 steps involved in peptide hydrolysis by chymotrypsin:
1. substrate binding: oxygen of -OH of chymotrypsin attacks carbonyl of substrate

2. nucleophilic attak of Ser-195 on peptide carbonyl group
3. collapse of tetrahedral intermediate
4. release of the amine component: protonated via His-57 when tetrahedral intermediate collapsed.
5. water binding (attacks nucleophile)
6. nucleophilic attack of water on the acyl-enzyme intermediate
7. collapse of the tetrahedral intermediate
8. release of the carboxylic acid (C-terminal) component.
What is the first step in peptide hydrolysis with chymotrypsin?
The substrate binds to chymotrypsin
In peptide hydrolysis with chymotrypsin, what happens after the substrate binds to the enzyme?
The oxygen atom of the side chain of serine 195 makes a nucleophilic attack on the carbonyl carbon atom of the target peptide: now there are four atoms bonded to the carbonyl carbon = tetrahedron (no longer planar).
-- this is an unstable intermediate with a negative charge on the oxygen derived from the carbonyl.
What happens after the chymotrypsin oxygen attack the carbonyl carbon of the target peptide during hydrolysis using chymotrypsin?
The tetrahedron intermediate collapses ⇉ generates acyl-enzyme.
-- faciliated by transfer of proton held by (+)His
In chymotrypsin hydrolysis of a peptide bond, what happens after the tetrahedron intermediate collapses?
The amine component now departs from the enzyme.
-- enzyme is now "aceylated"
In the chymotrypsin hydrolysis of a peptide, what happens after acetylation of the enzyme?
deacetylation
How does deacetylation (which occurs after acetylation of chymotrypsin in the hydrolysis of a peptide) occur?
It begins when a water molecule takes the plae occupied earlier by the amine component of the substrate.
What is the final step that readies the enzyme chymotrypsin for anotehr round of catalysis?
the release of carboxylic acid product.
Why is it the chymotrypsin only cleaves peptides bonds just past large, hydrophobic side chains?
Chymotrypsin has a large, hydrophobic pocket where these sidechains can fit into. Binding of the residue into this site positions the active site correctly for cleavage.
What does P1, P2, P3 refer to?
The residues on the amino-terminal side of the scissile bond (the bond to be cleaved), heading away from the scissile bond.
What does P1', P2', P3' refer to?
The residues on the carboxyl-terminal side of the scissile bond (the bond to be cleaved), heading away from the scissile bond.
What does S1, S2, S3 refer to?
The corresponding sites on the enzyme - correlating with P1, P2, P3 on amino-terminal side of scissile bond.
What do we call the bond to be cleaved?
Scissile bond
What is a scissile bond?
The bodn to be cleaved.
Where does the enzyme trypsin cleave peptide bonds?
after residues with long, positively charged side chains: arginine & lysine
Where does Elastase cleave peptide bonds?
after amino acids with small side chains, such as alanine and serine.
What enzyme cleaves bonds after long, positively charged side chains?
Trypsin
What enzyme cleaves peptide bonds after small residues?
Elastase
What enzyme cleaves peptide bonds after large, hydrophobic residues?
Chymotrypsin
What is in the S1 pocket of trypsin that accounts for it cleaving peptide bonds after long, positively charged side chains?
Asp 189 (-COO-): attracts and stabilizes a positively charged arginine or lysine residue in the substrate
What is in the S1 pocket of elastase that accounts for it cleaving peptide bonds after small side chains?
two valines: bulkier, therefore don't allow large residues to get into the pocket: enzyme isn't stable for cleavage unless residues are small.
What is the "catalytic triad"?
Three amino acids found in the active site of proteases: serine, aspartate, histidine
How can we evaluate a site-directed mutagenesis for subtilism (or another enzyme with the catalytic triad)?
Look at the comparitive log of Kcat/sec -- compare the rate of activity of mutated enzymes with the wild type and uncatalyzed (i.e. no enzyme present). Should tell us that mutations of any of the catalytic triad residues results in drastic reduction in Kcat
What residues make up the catalytic triad in chymotrypsin, and other homologs?
Asparagine; Histidine; SERINE
What residues make up the catalytic triad in esterases, and other homologs?
Asparagine; Histidine; CYSTEINE
How can we conclude that the catalytic triad is an especially effective approach for hydrolyzing peptide and related bonds?
Because it is found in many enzymes, even those with drastically different structure otherwise.
What residue(s) of an enzyme attack the carbonyl carbon in the hydrolysis of a peptide bond?
The catalytic triad. Particularly, the serine-histidine (or cysteine-histidine) pair act together to generate a nucleophile which does the attacking.
What is a cysteine protease?
An enzyme that uses a histidine-activated cysteine residue as the nucleophile for breaking peptide bonds.
What is an aspartyl protease?
An enzyme that uses an aspartate-activated water molecule as the nucleophile for breaking peptide bonds.
What is a metalloprotease?
An enzyme that uses a metal-activated water molecule as the nucleophile for breaking peptide bonds.
What are three classes of proteins that employ three alternative approaches to peptide-bond hydrolysis?
1. cysteine proteases
2. aspartyl proteases
3. metalloproteases.
What is the general strategy of proteases to break peptide bonds?
Generate a nucleophile that attacks the peptide carbonyl group.
How do cystein proteases work?
The histidine residue activates the cysteine, playing the role of nucleophile -- and attacks the carbonyl carbon of the peptide bond to break it.
How do aspartyl proteases work?
A pair of aspartic acid residues act together to allow a molecule of water to attack the peptide bond.
Asp 1 (deprotonated) - activates the attacking water molecule by poising it for deprotonation.
Asp 2 (protonated) - polarizes the peptide carbonyl group so that it is more susceptibe to attack.
What are some exampls of aspartyl proteases?
renin & pepsin
How do metalloproteases work?
the active site of the protein contains a bound metal ion (usually zinc) - activates a water molecule to act as a nucleophile and attack the peptide carbonyl group.
What are some examples of metalloproteases?
thermolysin & caboxypeptidase A
What are the 3 key features of cysteine/aspartyl/metallo-protease active sites?
1. activate a water molecule or another nucleophile

2. polarize the peptide carbonyl group

3. stabilize a tetrahedral intermediate
What are carbonic anhydrases?
Enzymes that increase the rate of reaction of CO₂ ⇄ HCO₃⁻ (carbon dioxide into bicarbonate ion; an already spontaneous reaction)
What do carbonic anhydrases do in the blood?
They convert (dehydrate)HCO₃⁻ to form CO₂. -- this is necessary for exhalation as the blood passes through the lungs.
What do carbonic anhydrases do in secretions (such as the aqueous humor of the eye)?
They convert CO₂ into HCO₃⁻
What does carbonic anhydrase do to CO₂?
Accelerates its hydration.
What are 3 key features of metals that make them useful in increasing chemical reactivity?
1. positive charge
2. ability to form strong yet kinetically labile bonds
3. capacity (often) to be in more than one oxidation state.
What tool is useful in giving us information about zinc sites in carbonic anhydrase?
X-ray crystallography
What is the effect of increasing pH on carbonic anhydrase Kcat (enzyme activity)?
Increasing the pH (ie more basic) increases activity. Kcat is greater at a greater pH
What state is zinc found in biological systems?
zinc is only found in the 2+ state! - it is always bound to 4 or more ligands.
In carbonic anhydrase, how is the zinc molecule bonded?
It is found in the 2+ state = must be bound to at least 4 ligands:
- 3 coordination sites are occupied by the imidazole ring of the 3 histidine residues
- 1 coordination site is occupied by a water molecule (or H+, depending on pH)
How does a zinc complex facilitate carbon dioxide hydration?
1. It facilitates the release of a proton of water molecule → OH⁻ (by binding to H2O which reduces the pKa of the H2O)

2. The CO2 binds to the enzyme's active site and it positioned to react with the OH⁻.

3. OH⁻ attacks CO2: converts it into HCO₃⁻

4. Catalytic site is regenerated with the release of HCO₃⁻ and the binding of another molecule of water.
What are the 4 steps of carbon dioxide hydration?
1. water deprotonation
2. carbon dioxide binding
3. nucleophilic attack of OH⁻ on CO2
4. displacement of bicarbonate ion by water
What does the binding of water to zinc do in the hydration of carbon dioxide?
It favors the formation of the transition state by facilitating proton release and by positioning the water molecule to be in close proximity to the other reactant.
What is the pKa of water bound to zinc?
8.7
What happens in the first step of the carbon dioxide hydration reaction?
The zinc-bound water molecule must lose a proton to regenerate the active form of the enzyme.
What limits the rate at which zinc-bound water molecules can lose a proton to regenerate active enzyme?
The rate of diffusion of the proton.
What is required for fast diffusion of protons from zinc-bound water molecules of an enzyme?
A buffer: can bind to or release protons.
How does increasing the [buffer] in a carbonic anhydrase reaction affect Kcat?
Increased [buffer] increases activity (Kcat) (curved, with flattening slope)
What does a proton shuttle do?
It allows the buffer components to participate in the reaction from solution (normally they are too big to react with active site of carbonic anhydrase)
What is the primary component of the proton shuttle of carbonic anhydrase II
Histidine 64
What does the his64 residue do as part of the proton shuttle in carbonic anhydrase II?
transfers protons from the zinc-bound water molecule to the protein surface, and then to the buffer
What are the 2 steps of the histidine proton shuttle?
1. His-64 abstracts a proton from the zinc-bound water molecule: generates a nucleophilic OH- and a protonated His

2. The buffer removes a proton from the His: regenerating the unprotonated form
What do restriction enzymes (restriction endonucleases) do?
Cleave DNA
What do restriction enzymes recognize to know that they should cleave foreign DNA?
recognition sequence / recognition site
Where do type II restriction enzymes work cleave?
Withing the recognition sequence
What is "cognate" DNA?
DNA molecules that contain recognition sites
What is a DNA molecule that contains a recognition sequence called?
Cognate DNA
What are the six mechanisms/principles of catalysis?
1. Covalent Catalysis
2. General Acid-Base Catalysis
3. Approximation
4. Orientation
5. Strain
6. Metal-Ion Catalysis
Generally, how does covalent catalysis work?
Active site contains a reactive group that is transiently modified
Generally, how does acid-base catalysis work?
Molecules otehr than H20 serve as proton donor/acceptor
Generally, how does approximation catalysis work?
Enzyme brings together two (or more) substrates to increase liklihood of reaction: increases effective [reactant]
Generally, how does orientation catalysis work?
Enzymes position molecules to react (nucleophilic attack) with specific stereochemistry
Generally, how does strain catalysis work?
Enzyme binds to substrate to induce strain: this facilitates transformation into transition state
Generally, how does metal-ion catalysis work?
* Metal facilitates formation of OH- nucleophile
* Metal serves as electrophile to stabilize (-)charges on reaction intermediates
* Metal can also coordinate/protect particular groups to insure they don't react
What is an affinity label?
A molecule that is similar in structure to a particular substrate for a specific enzyme -- works as an irreversible inhibitor by covalently modifying active site residues.
What type of enzyme is carbonic anhydrase?
Metalloenzyme. Catalyzes conversion of carbon dioxide and water into carbonic acids, protons, and bicarbonate ions.
What is the active site of most carbonic anhydrases?
A zinc ion (2+)
What is the reaction catalyzed by carbonic anhydrase in an environment with a lot of CO2 (such as in tissue)?
CO2 + H2O → H2CO3
What is the reaction catalyzed by carbonic anhydrase in an environment with not very much CO2 (such as the lungs)?
H2CO3 → CO2 + H2O
Who solved the 3D structure of chymtrypsin?
David Blow
What is the general structure of chymtrypsin?
Roughly spherical and comprises three polypeptide chains linked by disulfide bonds
What is the single polypeptide called that makes up chymotrypsin?
chymotrypsinogen: activated by proteolytic cleavage of the polypeptide to yield three chains
What is the number for transferases, which transfer functional groups between donor and acceptor molecules (ex: kinase)
2
What is enzyme category 2?
Transferases
What enzyme number are lyases, which Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds?
4
What number enzyme are hydrolases, which add water across a bond?
3
What does isozyme mean?
The individual members of a class of enzymes:
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyasazes
5. Isomerase
6. Ligase
What does carbonic anhydrase do?
In the blood, CO2 + water become carbonic acid. Carbonic anhydrase converts carbonic acid into bicarbonate ion in the blood so there isn't soo much acid around.

In the lungs, it turns the bicarbonate back into CO2 so that it can be exhaled.
What protects host DNA from degradation by endonucleases?
methylaaes: they methylate adenine bases within host recognition sequences. If a recognition sequence is methylated, it cannot be degraded by endonucleases.
What is a restriction-modification system?
A defense mechanism so that endonucleases don't cleave host DNA: the defense is made up of a restriction enzyme and a methylase, which methylates host DNA of correlating recognition sequence.