Biochemistry (9) - Catalytic Strategies

Front 1

What are the four classes of enzymes?

Back 1

1. serine proteases
2. carbonic anhydrases
3. restriction endonucleases
4. nucleoside monophosphate (NMP) kinases

Front 2

Which class(es) of enzymes require the addition of water to a substrate?

Back 2

1. serine proteases
2. carbonic anhydrases
3. restriction endonucleases

Front 3

Which class(es) of enzymes do require the prevention of water to a substrate?

Back 3

4. nucleoside monophosphate (NMP) kinase

Front 4

What is an example of a serine protease?

Back 4

chymotrypsin

Front 5

What is the "challange" of a serine protease (such as chymotrypsin)?

Back 5

promote a reaction that is almost immeasurably slow at neutral pH in the absence of a catalyst.

Front 6

What is the "challange" of carbonic anhydrase?

Back 6

To achieve a high absolute rate of reaction

Front 7

What is the "challange" of restriction endonucleases?

Back 7

Attaining a high degree of specificity

Front 8

What is the "challange" of NMP kinases?

Back 8

To transfer a phosphoryl group from ATP to a nucleotide and not to water.

Front 9

What are the four common strategies to catalyze specific reactions?

Back 9

1. covalent catalysis
2. general acid-base catalysis
3. catalysis by approximation
4. metal ion catalysis

Front 10

What is covalent catalysis?

Back 10

The active site contains a reactive groupe (usually powerful nucleophile) that becomes temporarily covalently attached to a part of the substrate during catalysis.

Front 11

How does general acid-base catalysis work?

Back 11

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.

Front 12

What is catalysis by approximation?

Back 12

Includes two distinct substrates.
NMP kinase: brings two nucleotides together to facilitate the transfer of a phosphoryl group from one nucleotide to another

Front 13

Who dow metal ion catalysis work?

Back 13

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.

Front 14

What do proteases do?

Back 14

They cleave proteins hydrolysis: addition of a molecule of water to a peptide bond.

Front 15

Why do we need proteases?

Back 15

Hydrolysis of peptide bonds are thermodynamically favored, but extremely slow. Proteases speed up the reaction from 100 years to milliseconds.

Front 16

What is responsible for the kinetic stability of peptide bonds?

Back 16

Resonance structure: gives planarity and structural stability (resistance to hydrolysis)

Front 17

What stregthens the carbon-nitrogen bond in a protein?

Back 17

Double bond character (resonance).Carbon is normally unreactive - needs an enzyme to facilitate nucleophilic attack of carbonyl group.

Front 18

Where does chymotrypsin cleave peptide bonds?

Back 18

Selectively on the carboxyl side of large hydrophobic amino acids:
- trp
- tyr
- phe
- met

Front 19

What type of catalysis does chymotrypsin utilize?

Back 19

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.

Front 20

What residue is extremely reactive in chymotrypsin?

Back 20

Serine residue (one of the twenty-eight) which lies in the active site.

Front 21

Does chymotrypsin obey M-M kinetics?

Back 21

Under steady-state, yes.

Front 22

What can proteases hydrolyze?

Back 22

Mostly amides, also esters (usually in vitro)

Front 23

What method can be used to moniter enzyme-substrate interaction?

Back 23

Stop-flow method

Front 24

What are the steps involved in chymotrypsin hydrolysis of peptide linkages?

Back 24

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.

Front 25

How can we see the two stages of cleavage by chymotrypsin?

Back 25

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.

Front 26

What is chymotrypsinogen?

Back 26

The name of the single polypeptide of chymotrypsin: chymotrypsin=3 of these linked by disulfide bonds

Front 27

List the 8 steps involved in peptide hydrolysis by chymotrypsin:

Back 27

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.

Front 28

What is the first step in peptide hydrolysis with chymotrypsin?

Back 28

The substrate binds to chymotrypsin

Front 29

In peptide hydrolysis with chymotrypsin, what happens after the substrate binds to the enzyme?

Back 29

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.

Front 30

What happens after the chymotrypsin oxygen attack the carbonyl carbon of the target peptide during hydrolysis using chymotrypsin?

Back 30

The tetrahedron intermediate collapses ⇉ generates acyl-enzyme.
-- faciliated by transfer of proton held by (+)His

Front 31

In chymotrypsin hydrolysis of a peptide bond, what happens after the tetrahedron intermediate collapses?

Back 31

The amine component now departs from the enzyme.
-- enzyme is now "aceylated"

Front 32

In the chymotrypsin hydrolysis of a peptide, what happens after acetylation of the enzyme?

Back 32

deacetylation

Front 33

How does deacetylation (which occurs after acetylation of chymotrypsin in the hydrolysis of a peptide) occur?

Back 33

It begins when a water molecule takes the plae occupied earlier by the amine component of the substrate.

Front 34

What is the final step that readies the enzyme chymotrypsin for anotehr round of catalysis?

Back 34

the release of carboxylic acid product.

Front 35

Why is it the chymotrypsin only cleaves peptides bonds just past large, hydrophobic side chains?

Back 35

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.

Front 36

What does P1, P2, P3 refer to?

Back 36

The residues on the amino-terminal side of the scissile bond (the bond to be cleaved), heading away from the scissile bond.

Front 37

What does P1', P2', P3' refer to?

Back 37

The residues on the carboxyl-terminal side of the scissile bond (the bond to be cleaved), heading away from the scissile bond.

Front 38

What does S1, S2, S3 refer to?

Back 38

The corresponding sites on the enzyme - correlating with P1, P2, P3 on amino-terminal side of scissile bond.

Front 39

What do we call the bond to be cleaved?

Back 39

Scissile bond

Front 40

What is a scissile bond?

Back 40

The bodn to be cleaved.

Front 41

Where does the enzyme trypsin cleave peptide bonds?

Back 41

after residues with long, positively charged side chains: arginine & lysine

Front 42

Where does Elastase cleave peptide bonds?

Back 42

after amino acids with small side chains, such as alanine and serine.

Front 43

What enzyme cleaves bonds after long, positively charged side chains?

Back 43

Trypsin

Front 44

What enzyme cleaves peptide bonds after small residues?

Back 44

Elastase

Front 45

What enzyme cleaves peptide bonds after large, hydrophobic residues?

Back 45

Chymotrypsin

Front 46

What is in the S1 pocket of trypsin that accounts for it cleaving peptide bonds after long, positively charged side chains?

Back 46

Asp 189 (-COO-): attracts and stabilizes a positively charged arginine or lysine residue in the substrate

Front 47

What is in the S1 pocket of elastase that accounts for it cleaving peptide bonds after small side chains?

Back 47

two valines: bulkier, therefore don't allow large residues to get into the pocket: enzyme isn't stable for cleavage unless residues are small.

Front 48

What is the "catalytic triad"?

Back 48

Three amino acids found in the active site of proteases: serine, aspartate, histidine

Front 49

How can we evaluate a site-directed mutagenesis for subtilism (or another enzyme with the catalytic triad)?

Back 49

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

Front 50

What residues make up the catalytic triad in chymotrypsin, and other homologs?

Back 50

Asparagine; Histidine; SERINE

Front 51

What residues make up the catalytic triad in esterases, and other homologs?

Back 51

Asparagine; Histidine; CYSTEINE

Front 52

How can we conclude that the catalytic triad is an especially effective approach for hydrolyzing peptide and related bonds?

Back 52

Because it is found in many enzymes, even those with drastically different structure otherwise.

Front 53

What residue(s) of an enzyme attack the carbonyl carbon in the hydrolysis of a peptide bond?

Back 53

The catalytic triad. Particularly, the serine-histidine (or cysteine-histidine) pair act together to generate a nucleophile which does the attacking.

Front 54

What is a cysteine protease?

Back 54

An enzyme that uses a histidine-activated cysteine residue as the nucleophile for breaking peptide bonds.

Front 55

What is an aspartyl protease?

Back 55

An enzyme that uses an aspartate-activated water molecule as the nucleophile for breaking peptide bonds.

Front 56

What is a metalloprotease?

Back 56

An enzyme that uses a metal-activated water molecule as the nucleophile for breaking peptide bonds.

Front 57

What are three classes of proteins that employ three alternative approaches to peptide-bond hydrolysis?

Back 57

1. cysteine proteases
2. aspartyl proteases
3. metalloproteases.

Front 58

What is the general strategy of proteases to break peptide bonds?

Back 58

Generate a nucleophile that attacks the peptide carbonyl group.

Front 59

How do cystein proteases work?

Back 59

The histidine residue activates the cysteine, playing the role of nucleophile -- and attacks the carbonyl carbon of the peptide bond to break it.

Front 60

How do aspartyl proteases work?

Back 60

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.

Front 61

What are some exampls of aspartyl proteases?

Back 61

renin & pepsin

Front 62

How do metalloproteases work?

Back 62

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.

Front 63

What are some examples of metalloproteases?

Back 63

thermolysin & caboxypeptidase A

Front 64

What are the 3 key features of cysteine/aspartyl/metallo-protease active sites?

Back 64

1. activate a water molecule or another nucleophile

2. polarize the peptide carbonyl group

3. stabilize a tetrahedral intermediate

Front 65

What are carbonic anhydrases?

Back 65

Enzymes that increase the rate of reaction of CO₂ ⇄ HCO₃⁻ (carbon dioxide into bicarbonate ion; an already spontaneous reaction)

Front 66

What do carbonic anhydrases do in the blood?

Back 66

They convert (dehydrate)HCO₃⁻ to form CO₂. -- this is necessary for exhalation as the blood passes through the lungs.

Front 67

What do carbonic anhydrases do in secretions (such as the aqueous humor of the eye)?

Back 67

They convert CO₂ into HCO₃⁻

Front 68

What does carbonic anhydrase do to CO₂?

Back 68

Accelerates its hydration.

Front 69

What are 3 key features of metals that make them useful in increasing chemical reactivity?

Back 69

1. positive charge
2. ability to form strong yet kinetically labile bonds
3. capacity (often) to be in more than one oxidation state.

Front 70

What tool is useful in giving us information about zinc sites in carbonic anhydrase?

Back 70

X-ray crystallography

Front 71

What is the effect of increasing pH on carbonic anhydrase Kcat (enzyme activity)?

Back 71

Increasing the pH (ie more basic) increases activity. Kcat is greater at a greater pH

Front 72

What state is zinc found in biological systems?

Back 72

zinc is only found in the 2+ state! - it is always bound to 4 or more ligands.

Front 73

In carbonic anhydrase, how is the zinc molecule bonded?

Back 73

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)

Front 74

How does a zinc complex facilitate carbon dioxide hydration?

Back 74

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.

Front 75

What are the 4 steps of carbon dioxide hydration?

Back 75

1. water deprotonation
2. carbon dioxide binding
3. nucleophilic attack of OH⁻ on CO2
4. displacement of bicarbonate ion by water

Front 76

What does the binding of water to zinc do in the hydration of carbon dioxide?

Back 76

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.

Front 77

What is the pKa of water bound to zinc?

Back 77

8.7

Front 78

What happens in the first step of the carbon dioxide hydration reaction?

Back 78

The zinc-bound water molecule must lose a proton to regenerate the active form of the enzyme.

Front 79

What limits the rate at which zinc-bound water molecules can lose a proton to regenerate active enzyme?

Back 79

The rate of diffusion of the proton.

Front 80

What is required for fast diffusion of protons from zinc-bound water molecules of an enzyme?

Back 80

A buffer: can bind to or release protons.

Front 81

How does increasing the [buffer] in a carbonic anhydrase reaction affect Kcat?

Back 81

Increased [buffer] increases activity (Kcat) (curved, with flattening slope)

Front 82

What does a proton shuttle do?

Back 82

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)

Front 83

What is the primary component of the proton shuttle of carbonic anhydrase II

Back 83

Histidine 64

Front 84

What does the his64 residue do as part of the proton shuttle in carbonic anhydrase II?

Back 84

transfers protons from the zinc-bound water molecule to the protein surface, and then to the buffer

Front 85

What are the 2 steps of the histidine proton shuttle?

Back 85

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

Front 86

What do restriction enzymes (restriction endonucleases) do?

Back 86

Cleave DNA

Front 87

What do restriction enzymes recognize to know that they should cleave foreign DNA?

Back 87

recognition sequence / recognition site

Front 88

Where do type II restriction enzymes work cleave?

Back 88

Withing the recognition sequence

Front 89

What is "cognate" DNA?

Back 89

DNA molecules that contain recognition sites

Front 90

What is a DNA molecule that contains a recognition sequence called?

Back 90

Cognate DNA

Front 91

What are the six mechanisms/principles of catalysis?

Back 91

1. Covalent Catalysis
2. General Acid-Base Catalysis
3. Approximation
4. Orientation
5. Strain
6. Metal-Ion Catalysis

Front 92

Generally, how does covalent catalysis work?

Back 92

Active site contains a reactive group that is transiently modified

Front 93

Generally, how does acid-base catalysis work?

Back 93

Molecules otehr than H20 serve as proton donor/acceptor

Front 94

Generally, how does approximation catalysis work?

Back 94

Enzyme brings together two (or more) substrates to increase liklihood of reaction: increases effective [reactant]

Front 95

Generally, how does orientation catalysis work?

Back 95

Enzymes position molecules to react (nucleophilic attack) with specific stereochemistry

Front 96

Generally, how does strain catalysis work?

Back 96

Enzyme binds to substrate to induce strain: this facilitates transformation into transition state

Front 97

Generally, how does metal-ion catalysis work?

Back 97

* 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

Front 98

What is an affinity label?

Back 98

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.

Front 99

What type of enzyme is carbonic anhydrase?

Back 99

Metalloenzyme. Catalyzes conversion of carbon dioxide and water into carbonic acids, protons, and bicarbonate ions.

Front 100

What is the active site of most carbonic anhydrases?

Back 100

A zinc ion (2+)

Front 101

What is the reaction catalyzed by carbonic anhydrase in an environment with a lot of CO2 (such as in tissue)?

Back 101

CO2 + H2O → H2CO3

Front 102

What is the reaction catalyzed by carbonic anhydrase in an environment with not very much CO2 (such as the lungs)?

Back 102

H2CO3 → CO2 + H2O

Front 103

Who solved the 3D structure of chymtrypsin?

Back 103

David Blow

Front 104

What is the general structure of chymtrypsin?

Back 104

Roughly spherical and comprises three polypeptide chains linked by disulfide bonds

Front 105

What is the single polypeptide called that makes up chymotrypsin?

Back 105

chymotrypsinogen: activated by proteolytic cleavage of the polypeptide to yield three chains

Front 106

What is the number for transferases, which transfer functional groups between donor and acceptor molecules (ex: kinase)

Back 106

2

Front 107

What is enzyme category 2?

Back 107

Transferases

Front 108

What enzyme number are lyases, which Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds?

Back 108

4

Front 109

What number enzyme are hydrolases, which add water across a bond?

Back 109

3

Front 110

What does isozyme mean?

Back 110

The individual members of a class of enzymes:
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyasazes
5. Isomerase
6. Ligase

Front 111

What does carbonic anhydrase do?

Back 111

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.

Front 112

What protects host DNA from degradation by endonucleases?

Back 112

methylaaes: they methylate adenine bases within host recognition sequences. If a recognition sequence is methylated, it cannot be degraded by endonucleases.

Front 113

What is a restriction-modification system?

Back 113

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.