Enzymes

Question 1

The first class of enzymes is _______________, which catalyzes electron transfer reactions, and an example is alcohol dehydrogenase.

Answer 1

Oxidoreductase

Question 2

The second class of enzymes is ______________, which catalyzes group transfer reactions, and DNA polymerase is an example of one.

Answer 2

Transferase

Question 3

The third class of enzymes is ___________________, which catalyzes hydrolysis reactions, and examples include chymotrypsin and RNase A.

Answer 3

Hydrolyase

Question 4

The fourth class of enzymes is ____________, which catalyzes cleavage reactions or eliminations, and carbonic anhydrase is an example.

Answer 4

Lyase

Question 5

The fifth class of enzyme is ______________, which catalyzes the transfer of groups within a molecule to yield isomers, and phosphohexose isomerase is an example.

Answer 5

Isomerase

Question 6

The sixth and last class of enzymes is _____, which catalyzes the formation of covalent bonds by condensation reactions and ATP, and aminoacyl-tRNA synthetase is an example.

Answer 6

Ligase

Question 7

There are four mechanisms in which an enzyme can catalyze a reaction. What are they?

Answer 7

1. Electrostatic catalysis
2. Proximity and orientation effects
3. General acid/base catalysis
4. Covalent or nucleophilic catalysis

Question 8

_______________ catalysis involves charged groups in an active site that help stabilize the transition-state; the active sites are generally hydrophobic and exclude water, thereby enabling the reaction to occur at a low dielectric constant, which increases the strength of charged interactions.

Answer 8

Electrostatic

Question 9

In electrostatic catalysis, pKa values can ______________ from tabulated values.

Answer 9

Differ greatly

Question 10

An example of electrostatic catalysis discussed in class is the deprotonation of aspartate. When in the presence of ___________, a positively charged amino acid, aspartate is ______________ (more or less) likely to deprotonate, thereby _____________ the pKa.

Answer 10

Lysine

More likely

Lowering

Question 11

In proximity and orientation effects, the enzyme brings the catalytic groups together, holding the enzyme and substrate in ______________________ thereby freezing _______________ and _____________ motion of the substrate and catalytic groups.

Answer 11

Proper orientation

Translational

Rotational

Question 12

Proximity and orientation effects is based upon __________________, the theory that a reaction can only proceed when molecules are in the proper orientation and energy level when they collide.

Answer 12

Collision theory

Question 13

_______________________ refers to proton transfers mediated by _____ acids and bases.

Answer 13

General acid/base catalysis

Weak acids and bases

Question 14

General __________ catalysis involves catalysis where protein transfers from an acid occur.

Answer 14

Acid

Question 15

General _______ catalysis involves catalysis where proton abstraction from a base occurs.

Answer 15

Base

Question 16

What enzyme did we use in class that functioned as general acid/base catalyst?

Answer 16

RNase A, which catalyzes the hydrolysis of RNA of its sugar-phosphate backbone

Question 17

In the acid/base catalysis of RNase A, two essential ___________ residues are required in the active site: _______12 and ______119.

Answer 17

Histidine

His12

His119

Question 18

(Re RNase A)

First, His___ functions as a base and abstracts a proton from the hydroxyl group from the ___ (carbon) of ribose. This enables the oxygen atom from the hydroxyl group to function as a better _______________, allowing for its attack of the electrophilic ________ atom in the sugar-phosphate backbone. An unstable intermediate forms, requiring that the oxygen in the ____________ group “kick back” to break the phosphodiester bond. To do so, His___ must function as an acid, making for a better leaving group.

Answer 18

His12

C2’

Nucleophile

Phosphate

O = P

His119

Question 19

(Re RNase A)

Ultimately, this results in the loss of a nucleoside and formation of an unstable intermediate. To regenerate the enzyme, His___ now functions as a base, abstracting a proton from water. The resulting hydroxide ion makes for a better _______________ and attacks the electrophilic phosphate. Ultimately, an unstable penta-intermediate forms, oxygen “kicks back,” and expels the ___ (carbon) oxygen on the ribose. This requires that the oxygen abstract a proteon from His___, which now functions as a _________.

Answer 19

His119

Nucleophile

C2’

His12

Acid

Question 20

Make sure you understand the mechanism of RNase A (a hydrolase).

Answer 20

Question 21

From the list of amino acids, which could be used as general acid catalysts?

Answer 21

Glutamic acid (E)

Aspartic acid (D)

Histidine (H)

Arginine (R)

Lysine (K)

Serine (S)

Cysteine (C)

Tyrosine (Y)

Question 22

Under what conditions might the amino acids listed as acids NOT act as general acid catalysts?

Answer 22

When they are deprotonated, so when pH > pKas

Question 23

From the list of amino acids, which could be used as general base catalysts?

Answer 23

Glutamic acid (E)

Aspartic acid (D)

Histidine (H)

Arginine (R)

Lysine (K)

Serine (S)

Cysteine (C)

Tyrosine (Y)

Question 24

Under what conditions might the amino acids listed above NOT function as general base catalysts?

Answer 24

When protonated; pH < pKa

Question 25

From the list of amino acids, which might serve as nucleophiles and under what specific conditions?

Answer 25

Serine (S) Cysteine (C) Tyrosine (Y) Lysine (K) Histidine (H) Arginine (R) Glutamic acid (E) Aspartic acid (D) When they are deprotonated; pH \> pKa

Question 26

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ catalysis involves the transient formation of a covalent bond between the enzyme and substrate.

Answer 26

Covalent or nucleophilic catalysis

Question 27

Be familiar with the general mechanism of covalent or nucleophilic catalysis.

Answer 27

1. Nucleophilic attack by a group on the enzyme that forms a covalent bond between E and S 2. Stabilization of transition state by amino acids in the active site of the enzyme 3. Regeneration of enzyme

Question 28

A type of covalent/nucleophilic catalysis we discussed in class was \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_, another hydrolase.

Answer 28

Chymotrypsin

Question 29

Chymotrypsin is a ______ protease because its catalytic mechanism involves a _______ acting as a nucleophile. In addition, two other amino acids, histidine and \_\_\_\_\_\_\_\_\_\_, serve important roles.

Answer 29

Serine Serine Aspartic acid

Question 30

The catalytic triad of chymotrypsin, like all serine proteases, consists of Ser195, His57, and \_\_\_\_102.

Answer 30

Asp102

Question 31

The catalytic traid perfectly aligns, and then a ___________ pocket forms around the oxygen atom.

Answer 31

Hydrophobic

Question 32

Chyotrypsin cleaves on the C-terminal sites of aromatic amino acids: \_\_\_\_\_\_\_\_\_, \_\_\_\_\_\_\_\_\_\_\_\_, and \_\_\_\_\_\_\_\_\_\_\_.

Answer 32

W, F, and Y

Question 33

The oxyanion hole stabilizes the ___________________ over the substrate.

Answer 33

Transition state

Question 34

In the chymotrypsin catalysis, His\_\_\_ functions as a general base, abstracting a proton from Ser\_\_\_. Asp\_\_\_ hydrogen bonds with His\_\_\_, making it a better base. Ser195 acts as a nucleophile, attacking the carbonyl carbon to the _______ (left or right) of an aromatic R group. A tetrahedral intermediate forms, which is unstable; however, the oxyanion moves into the _________ hole, which stabilizes the charge and the transition state. Oxygen "kicks back," and expels the amino end after His57 functions as an \_\_\_\_\_, donating its proton to enable the loss of a better leaving group. A product leaves, and the enzyme-substrate _____________ complex is formed. His57 abstracts a proton from \_\_\_\_\_\_\_\_, which then functions as a nucleophile, attacking the remaining carbonyl carbon. Again, a tetrahedral intermediate forms, and the oxyanion moves into the __________ hole, stabilizing the transition state. Oxygen kicks back and expels the enzyme as it abstracts a proton from His\_\_\_\_\_. This regenerates the enzyme and results in the second product.

Answer 34

His57 Ser195 Asp102 His57 Right Oxyanion hole Acid Enzyme-substrate covalent complex Water Oxyanion hole His57

Question 35

You must be familiar with the mechanism of covalent/nucleophilic catalysis of a serine protease (i.e., chymotyrpsin, trysin, which cleaves after R and K).

Answer 35

Ser195 His57 Asp102 (vs. in general acid/base catalysis RE: RNase A - His12 & His119)

Question 36

How was the mechanism of chymotrypsin elucidated?

Answer 36

Kinetic studies in which p-Nitrophenol was reacted with chymotrypsin

Question 37

What is the "fast" step in the chymotrypsin mechanism?

Answer 37

The first acylation where Ser195 nucleophilically attacks the substrate

Question 38

What is the "slow" step in the chymotrypsin mechanism?

Answer 38

The second deacylation step in which water cleaves the ES covalent complex, reforming Ser195 and forming the second product

Question 39

The enzymatic action of chymotrypsin involves two substrates and is thus called a _________ reaction. These reactions can occur by single displacements or double displacements. ___________ reactions occur by double displacement, and they are often described using Cleland notation.

Answer 39

Bisubstrate Ping Pong reactions

Question 40

Trypsin and elastase are homologs of chymotrypsin. If each of these proteases cleave peptide bonds in the same way, what dictates the specificity of each?

Answer 40

Differences in the protease pockets

Question 41

Chymotrypsin binds substrates based on _____________ pocket. Trypsin binds substrates based on ____________ pocket.

Answer 41

Hydrophobic Negatively charged

Question 42

Chymotrypsin works in the small intestine to aid in digestion of proteins. It is synthesized in the pancreas in the form of a zymogen. What is a zymogen?

Answer 42

An inactive protein

Question 43

Why is the formation of chymotrypsin as a zymogen important?

Question 44

The formation of zymogens is a method of ___________ enzyme activity.

Answer 44

Regulating

Question 45

\_\_\_\_\_\_\_\_\_\_\_\_\_ proteins can also regulate enzyme activity.

Answer 45

Inhibitor

Question 46

An example of oxidoreductase is \_\_\_\_\_\_\_\_\_\_\_\_\_\_.

Answer 46

Alcohol dehydrogenase

Question 47

REDOX reactions require enzymes with \_\_\_\_\_\_\_\_\_\_\_. Many use nicotinamid adenine dinucleotide (NAD⁺). Alcohol dehydrogenase is one such enzyme. In the mechanism, electrons move in the form of the hydride ion (:H^-). Ethanol + NAD⁺ --\> Acetylaldehyde + NADH + H⁺

Answer 47

Cofactors

Question 48

Ethanol + NAD+ --\> Acetylaldehyde + NADH + H+ Ethanol is \_\_\_\_\_\_\_\_\_\_\_\_\_. NAD+ is \_\_\_\_\_\_\_\_\_\_\_. Therefore, NAD+ is the _______________ agent, and ethanol is the ______________ agent.

Answer 48

Oxidized Reduced Oxidizing agent Reducing agent

Question 49

What is the important point about oxidoreductase mechanism?

Answer 49

Zn2+ stabilizes the negative charge on oxygen of substrate to facilitate the reaction

Question 50

When a metal ion is used to help in enzymatic mechanicms, the catalysis is called __________________ catalysis.

Answer 50

Metal-ion

Question 51

Be sure to understand the mechanism of alcohol dehydrogenase, an oxidoreductase

Answer 51

1. Zn2+ sits in active site 2. Base deprotonates ethanol (alcohol), and Zn2+ stabilizes negative charge 3. Negative charge kicks back onto carbon, ultimately releasing a hydride ion (:H-), which is extremely reactive 4. The hydride attacks the coenzyme's aromatic ring system 5. Another hydrogen is attacked to the coenzyme, here NAD+, with appropriate shifts in ring structure

Question 52

DNA polymerase is an enzyme _____________ that catalyzes a phosphoryl group transfer reaction as it synthesizes DNA. The enzyme requires two Mg2+ ions in the active site; therefore, it is an example of _____________ catalysis.

Answer 52

Transferase Metal-ion catalysis

Question 53

The role of Mg2+ in DNA polymerase mechanism is to stabilize the negative charge on the phosphate groups and to stabilize the negative charge that results from _______________ attack of the C3' __________ group.

Answer 53

Nucleophilic attack Hydroxyl

Question 54

An example of a lysase is \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_. It is an enzyme that catalyzes te rapid conversion of Co2 and H20 to bicarbonate and a proton in aqueous solution. It is classified as a _____________ because it contains a tightly bound metal ion cofactor that is essential for the reaction.

Answer 54

Carbonic anhydrase Metalloenzyme

Question 55

The metalloenzyme of carbonic anhydrase is \_\_\_\_\_\_\_\_.

Answer 55

Zn2+

Question 56

The role of Zn2+ in carbonic anhydrase is to facilitate the acidification of water so that a proton can leave, providing the source of ____________ needed for the reaction to occur.

Answer 56

Hydroxide

Question 57

CO2 + H20 --\> HCO3- + H+ This reaction is catalyzed by...?

Answer 57

Carbonic anhydrase, a lysase

Question 58

Three invariant histidines are required in the metal-ion catalysis with carbonic anhydrase. Be familiar with the mechanism.

Answer 58

Question 59

An example of an isomerase we explored in class was phosphohexose isomerase, which catalyzes the *reversible isomerization* of glucose-6-phosphate, a aldehyde monosaccharide, to \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_, a ketone monosaccharide. G6P \<----\> F6P

Answer 59

Fructose-6-phosphate 1. A base deprotonates C2 2. A double bond forms between C2 and C1 3. Oxygen from the carbonyl of C1 abstracts a protein from an acid 4. A base deprotonates the C2 hydroxyl group 5. The double bond between C2 and C1 abstracts a proton from an acid

Question 60

An example of a ligase is aminoacyl-tRNA synthetase. It catalyzes the esterification of an amino acid to its corresponding tRNA. The amino acid is attached to the 3' OH of the terminal amino acid via ester linkage. Most organisms have an amino-acyl tRNA synthetase for each amino acid 1. An enzyme-bound intermediate (aminoacyl-AMP) is formed 2. The aminoacyl group is transferred to its specific tRNA

Answer 60

Question 61

The reaction catalyzed by aminoacyl-tRNA synthetase is amino acid + tRNA + ATP --\> amino-acyl-tRNA + PPi + Amp --\> 2Pi The formation of inorganic phosphate from pyrrophosphate is the driving force of the reaction