Chapter 11: Enzymatic Catalysis

Question 1

How do enzymes differ from chemical catalysts?

Answer 1

1) higher reaction rates- enzyme rates are 10^12 times faster than an uncatalyzed rxn
2) milder rxn conditions- some chemical catalysts need intense heat, acidic conditions, etc. Becuse enzymes are biological, they need to be able to function in relatively neutral temperatures and physiological ph.
3) Greater rxn specificity
4) capacity for regulation- an enzyme can be affected by allosteric effectors, covalent modifications, substrate concentrations etc.
5) Enzymes are protein based, but chemical catalysts can be made of almost anything- may be just ions, etc.

Question 2

How are enzymes classified?

Answer 2

via Enzyme Commission number classified into 6 major classes, sub class, sub-sub classes, and the serial number. Denoted by 4 general numbers.

Question 3

6 Major classes of enzymes

Answer 3

1) oxidoreductase- catalyzes reactions using oxidation-reduction catalysis.
2) transferases: transfer of functional group reaction catalysis.
3) hydrolasys: hydrolysis rxn catalysis
4) lyases: group elimination of double bonds
5) Isomerases; helps rxn proceed via isomerization, changing structure of compound but keeping same molecular equation
6) Ligases: bond formation coupled with atp hydrolysis

Question 4

Of the six major classes of enzymes, which mechanism uses ATP hydrolysis coupling

Answer 4

Ligases- involves bond formation and therefore they need energy

Question 5

Generally, what kind of complimentarity do enzymes exhibit?

Answer 5

1) geometric complimentarity: the site consists of an indentation on the surface of an enzyme that is complementary in the shape of the substrate
2) electronic complementarity: amino acids that form the binding site arranged to actually attract the substrate.

Question 6

Define induced fit

Answer 6

Upon substrate binding the enzyme-substrate complex undergoes conformational change.

Question 7

How does the active site play a role in catalysis in terms of G?

Answer 7

provides an energetically favorable environment.

Question 8

T/F: Enzyme affects positional equilibrium by allowing my products to be produced in a shorter amount of time.

Answer 8

False: enzymes do not contribute to equilibrium position. The help speed up the rate to get TO equilibrium. K remains the same.

Question 9

T/F: true proteins contain a mixture of D and L-enantiomers of amino acids.

Answer 9

False. True proteins only contain L-amino acids. If they contain a mixture of D and L amino acids, they most likely end up carrying nucleic acids too and are just a giant biological compound.

Question 10

T/F: enzymes are chiral

Answer 10

True, all enzymes are chiral.

Question 11

Define cofactor

Answer 11

Any factor required for enzyme activity of protein function. Can be inorganic or an organic/metalorganic molecule

Question 12

Term for an organic cofactor

Answer 12

a coenzyme

Question 13

What is a coenzyme that is only associated with the enzyme part of the time?

Answer 13

a co substrate.

Question 14

Example of a cosubstrate

Answer 14

NAD+ and NADP+ are cosubstrates. NAD+ is an obligatory oxidizing agent in alcohol dehydrogenase rxn. the product NADH dissociates from the enzyme for reoxidation to NAD+.

Question 15

What is a coenzyme that is associated with the enzyme all the time?

Answer 15

a prosthetic group

Question 16

Name an example of a prothetic group

Answer 16

Heme prosthetic group are tightly bound to cytochrome proteins through hydrophobic and Hbonding interactions and covalent bonds between heme and protein sidechains.

Question 17

What is a holoenzyme

Answer 17

a catalytically active enzyme-cofactor complex

Question 18

Apoenzyme

Answer 18

an enzymatically inactive protein resulting from the removal of the holo enzyme cofactor.

generally: apoenzyme + cofactor –> holoenzyme

Question 19

Do cofactors get used up in a reaction even though the enzyme gets reformed?

Answer 19

co factors do get used up but then they reform in order to complete the catalytic cycle

Question 20

two requirements in order for particle collisions to result in a reaction:

Answer 20

1) correct orientation

2) reactants must possess a minimum energy (activation energy) to initiate the chemical rxn.

Question 21

the symbol for activation energy

Answer 21

deltaG with dash through equals sign

Question 22

At higher temperatures, more molecules possess the ____ to overcome activation energy, and thus the reaction rate increases

Answer 22

kinetic energy

Question 23

deltaG=/= or K constant is manipulated by an enzyme

Answer 23

Delta G is lowered by an enzyme and allows the activation energy to be more easily overcome. K is the equilibrium constant and is thus not affected because enzymes do not contribute to equilibrium shifts

Question 24

What is the Arrhenius equation used for?

Answer 24

brings activation energy, collision frequency and orientation to determine either equilibrium constant (K) or activation energy

Question 25

T/F The equilibrium constant K is related to overall delta G and not to the activation energy barrier dG=/=

Answer 25

True. dG=/= affects RATE (enzymatically manipulated), dG affects equilibrium( Keq)

Question 26

The greater the value of dG=/=, the _____ rxn

Answer 26

the slower the reaction. Having a higher dG=/= menas that it will take more time or that there will be less reactant molecules that possess enough energy to be able to over come the reaction barrier.

Question 27

T/F The dG=/= can be reduced by catalyst, but dG stays the same

Answer 27

True, dG is not affected by enzyme because it is an indicator of equilibrium. dG=/= can be reduced by catalyst to allow the reaction to approach equilibrium faster

Question 28

define rate enhancement

Answer 28

the efficiency of a catalyst, the difference between the values of dG=/= in the catalyzed and uncatalyzed rxn.

Question 29

methods that enzymes increase the rxn rate

Answer 29

1) increase the frequency of molecule reaction
2) increase the likelihood that the reaction collides with the optimal orientation for rxn.
3) increase the number of reactants with the energy to overcome the dG=/= by increasing the KE of reactants (increase temp), or you could decrease dG=/= to allow more reactants to possess enough energy to proceed through the rxn.

Question 30

What type of catalytic mechanism increases the reaction rate if the orientation of the substrate is the most optimal/

Answer 30

Proximity and orientation effects; a mechanism that increases the odds that particles will occur in the correct orientation for reaction.

Question 31

Define electrostatic catalysis

Answer 31

charged groups may help stabilize transition state of the reaction

Question 32

Define preferential binding of substrate

Answer 32

when an enzyme binds the transition state of the reaction with greater affinity that its substrates or products

Question 33

Define transition state analog

Answer 33

stable molecule that geometrically and electronically resembles the transition state, and thus acts as an inhibitor of the enzyme.

Question 34

explain concerted general acid-base catalysis

Answer 34

process by which a proton is removed by a bronsted base from a bronsted acid to lower the free energy of the reactions transition state

Question 35

In RNaseA system, which are the two amino acids that are involved in acid base catalysis?

Answer 35

His12 and His119

Question 36

Explain the mechanism of RNA hydrolysis via RNaseA

Answer 36

His12 first acts as a general BASE and extracts a proton from the 2’OH group of the RNA molecule, promoting oxygen’s nucleophillic attack on the adjacent phosphorous atom. Simultaneously, His119 acts as a general ACID and donates a proton to the RNA leaving group, cleaving the bond and releasing one RNA. As the leaving group is extracted, a 2’3’cyclic intermediate is formed, and water enters the active site. His12 now acts a general ACID and his119 acts as a general BASE to promote hydrolysis of the intermediate and release the other RNA molecule. The enzyme active site is now back to the original conformation, with His119 active group being protonated and His12 “nitrogen active group” being deprotonated.

Question 37

What’re the problems with RNA hydrolysis that RNAse A aid in?

Answer 37

1) 2’ Hydroxyl is relatively stable and is a bad nucleophile
Solution: His12 acts as a general base and extracts OH’s proton so it can nucleophillically attack the phosphorous

2) RO- at end of enzyme is a bad leaving group
Solution: His119 donates a proton and acts as a general acid to the leaving group (RNA) to improve the quality of the LG.

Question 38

What is the role of His119 in RNA hydrolysis?

Answer 38

His119 improves the quality of the leaving group by first acting as an acid and then returns enzyme to original state by acting as a base and re-protonating itself using water.

Question 39

Explain covalent catalysis

Answer 39

accelerates the reaction rate through transient formation of a catalyst-substrate covalent bond. Covalent bond is usually formed by reaction of a nucleophillic group on the catalyst and an electrophillic group on the substrate.

Question 40

Explain a general schiff base mechanism in acetoacetate-acetone

Answer 40

Uses covalent catalysis. The schiff base forms a transient covalent bond with the acetoacetate substrate, with the nitrogen of the schiff base being positively charged. The positive charge acts as an electrophile and draws away electrons from the negatively charged carboxylate group from the acetate substrate, seeding up the reaction. The schiff base is eliminated by the reverse reaction, rendering acetone.

Question 41

How does a schiff base help speed up the reaction of acetoactate to form acetone?

Answer 41

Positive charge on the nitrogen acts as an electron sink, unlike the uncatalyzed reaction, which renders an enol/enolate, that usually undergoes a time consuming resonance stabilizing mechanism involving negative carbon to neutral carbon process. The positive charge of schiff base that is created upon bonding the substrate to the amine catalyst sucks the electron away from negative carboxylate, speeding up the reaction (withdraws the electrons from the reaction center (substrate) by the now electrophilic catalyst.

Question 42

T/F nucleophilicity is closely related to basicity

Answer 42

True

Question 43

What 3 roles do metal ion cofactors aid in catalysis?

Answer 43

1) bind to substrates to orient them properly for reaction
2) mediate oxygen-reduction reactions through reversible changes in the metal ion’s oxidation state
3) by electrostatically stabilizing/shielding negative charges

Question 44

What is an advantage to using metal ion cofactors as a form of catalysis instead of acid-base catalysis?

Answer 44

metal ions essentially can act as protons but are good for biological systems because an increase in conc. of metal ions can be present at neutral pH whereas you cannot increase the proton concentration without changing the ph level, which is unfavourable to a biological organism.

Question 45

Describe electrostatic catalysis

Answer 45

Catalysis through the use of favourable electrostatic interactions, usually referring to ionic interactions. Stabilized developing charge in the transition state and helps orientate the substrate for optimal reaction.

Uses aformentioned aspects of catalysis, such as orientation/proximity effects, preferential stabilization of T state, acid/base catalysis and metal ion catalysis.

Question 46

Generally, what is a serine protease?

Answer 46

an enzyme that contains a reactive Ser residue in its active site that helps hydrolyze peptide bonds (protease)

Question 47

What secondary structure form does chymotripsin and trypsin proteins fold into?

Answer 47

2 domains are folded into beta pleated sheets.

Question 48

What three amino acids make up the catalytic triad? how do they bond together?

Answer 48

His57, Ser195 and Asp102 make up the catalytic triad and are located on the active site held together by hydrogen bonds

Question 49

what is the substrate specificity of chymotrypsin? What other important amino acids are in the chymotrypsin active site?

Answer 49

bulky aromatic side chains such as Trp, Tyr, Phe etc. can fit into the hydrophobic pocket can fit into the active site.

Gly226, Ser189, Gly216.

Question 50

what is the substrate specificity of trypsin? What other important amino acids are in the trypsin active site?

Answer 50

trypsin tends to target positively charged amino acids such as lysine or arginine because it contains negatively charged aspartate residue, which would allow it to form ion pairs.

Gly226,Arg189, Gly216

Question 51

What is the substrate specificity of elastase? What other important amino acids are in the trypsin active elastase?

Answer 51

can hydrolyze elastin, which is rich in valine, alanine and glycine, particularly cleaving proteins at the site of the alanine residue. Instead of having Gly226 and GLy 216 in their active site, they have Val226 and Thr216

Question 52

Describe the general mechanism of chymotripsin

Answer 52

His57 undergoes functions as a general base by extracting a proton from ser 195 to make it a better nucleophile.
Ser195 then nucleophilically attacks the sessile peptide bond via COVALENT CATALYSIS to form a tetrahedral intermediate
The formation of the tetrahedral intermediate forms an oxyanion hole near ser195 and the carbonyl group (c terminus) end of the peptide, drawing the c terminal anion closer into the active site. The oxianion hole then promotes transition state stability by allowing the c terminus anion of the peptide to form hydrogen bonds with glycine in the active site. (PREFERENTIAL BINDING OF THE TRANSITION STATE)
The tetrahedral intermediate is broken down and the bond between the c terminus and n terminus of the peptide is cleaved because the carbonyl anion acted as a nucleophile, forming a double bond at the carbon. Asp102 acts as a polarizing group (ELECTROSTATIC CATALYSIS), and His57 acts as a general acid to protonate the n terminal end, letting RNH2 leave as a leaving group.

As the amine product is released, water from the solvent fills the active site (HYDROLYTIC CLEAVAGE) and His57 acts as a general base again, deprotonating the water, forming a good nucleophile. The now -OH complex nucleophilically attacks the remaining carbonyl terminal end of the peptide to form a new tetrahedral intermediate, (carbonyl and OH portion of water).
His57 then acts as a general acid to break down the tetrahedral intermediate into a carbonyl product as it donates its proton to ser195 in order to restore its OH group of its side chain. This yields a free enzyme and an r-COOH product.

Therefore: Covalent catalysis+general base, general acid catalysis, hydrolytic cleavage, general base catalysis and general acid catalysis

Question 53

How does the oxyanion hole aid in chymotripsin catalysis

Answer 53

Oxyanion hole is in the pocket of chymotrypsin’s active site that stabilizes the transition state’s negative charge on the carbonyl oxygen by allowing it t move deeper into the active site, forming two hydrogen bonds with ser195 and glycine.

It aids in catalysis by lowering the activation energy because the transition state stability is increased, which promotes catalysis. the preferential binding to the ts (tetrahedral) over the ES complex is responsible for serine proteases catalytic efficiency because there are more favourable hydrogen bonds occurring

Question 54

What are the obstacles that serine proteases need to overcome when hydrolyzing poly peptides?

Answer 54

1) recognizing specificity pocket
2) H2O is a bad nucleophile.
Solution: Via general base catalysis, the quality of the water nucleophile is improved because his 57 steals a proton, while the oxygen attaches to the carbonyl portion of the peptide, forming the second tetrahedral intermediate
3) tetrahedral intermediate is unstable because of the distortion caused by peptide moving deeper into the active site
solution: the oxyanion hole near the ser195 residue allows the carbonyl anion to get stabilized by hydrogen bond formation.
4) R-NH- is a bad leaving group.
Solution: His 57 acts as a general acid and protonate the leaving group to make it R-NH2

Question 55

What is a zymogen

Answer 55

the inactive form of a serine protease/ digestive enzyme

Question 56

How does the mechanism of tripsinogen to tripsin activate the zymogen?

Answer 56

the mechanism causes change in the catalytic triad and active site of tripsin in order to activate it

Question 57

Explain the mechanism of how tripsin in activated

Answer 57

tripsinogen gets activated by an enteropeptidase that excises the N terminal portion from tripsinogen by cleaving its Lys15-Ile16 peptide bond.
Enterostripsin clips C-terminal of Lys and thus creates a new N terminus of Ile.

Ile is hydrophobic and non polar, and moves into the interior of the protein where it forms salt bridges with asp194, which is near tripsins catalytic triad. The salt bridges cause conformational change of the catalytic triad and the active site, making tripsin now ready for catalysis.