topic 05 enzyme catalysis

Question 1

define substrate

Answer 1

molecule on which an enzyme acts

Question 2

the names of enzymes often end with?

Answer 2

-ase

-in

-zyme

Question 3

what is special about enzymes?

Answer 3

specificity: will recognize one/few molecules out of thousands

accurate/fidelity: almost never makes mistakes. resulting in almost a 100% yield

rapidity: accelerates reactions greatly

ability to work under mild conditions: such as low temperatures/pressures, neutral pH, etc - however, they do work best under specific conditions

regenerate: they are regenerated during the reaction

Question 4

describe the active site of an enzyme

Answer 4

cleft or crevice between domains

Question 5

define transition state

Answer 5

the state, in a chemical reaction, in which it has the highest energy. once this state has been achieved, the reaction is irreversible

it is the intermediate form between reactants and products

Question 6

what must reactants over come to form products? (what’s the symbol)

Answer 6

energy barrier: ∆G≠

Question 7

what are the mechanisms enzymes employ to lower the activation energy? which method is the most common?

Answer 7

A) enzyme binds to 2 substrate molecules and orient them in a way to encourage a reaction

B) binding of substrate to enzyme rearranges the electrons in the substrate, creating partial negative and positive charges that encourage a reaction

most common method
C) enzyme strains the bound substrate molecule, forcing it toward a transition state to encourage a reaction

Question 8

define transition state analogues. give example

Answer 8

enzyme inhibitors that resemble transition states but are not reactive

many drugs and antibiotics are inhibitors

Question 9

describe enzyme cofactors. what are some enzyme cofactors (what form do they come in/what do they consist of)?

Answer 9

a required partner that some enzymes (apoenzymes) require to function. they may be essential but they also increase the rate of reaction

enzyme cofactors could be proteins, metal ions, or co-enzymes (organic/metallo-organic molecules derived from vitamins). most consist of protein or RNA or a complex of RNA and protein

Question 10

define apoenzymes

Answer 10

an enzymatically inactive protein part of an enzyme that requires a cofactor for its activity

Question 11

what do enzymes not affect?

Answer 11

equilibrium constant K: the ratio of substrate to products

different in free energy between reactants and products (∆G), this is because ∆G is related to K

Question 12

what has the highest affinity for a reaction’s transition state?

Answer 12

enzymes
…

NOT substrates or products

Question 13

what are the strategies used by enzymes to attain transition states?

Answer 13

bringing substrates together (in multi-substrate reaction)

orientating substrates in a favourable geometry

supplying proton acceptors/donors & electron donors/acceptors

excluding water (in some cases)

stressing substrate physically or electronically

selective binding of the transition state

Question 14

what may happen to enzymes under extreme conditions such as pH and temperatures?

Answer 14

extreme pH
enzymes may be protonated/deprotonated or may denature

extreme temperature
enzymes may become unstable due to increasing reaction rate

Question 15

what is the catalytic triad of serine protease made up of?

Answer 15

Serine, Aspartate, and Histidine

Question 16

what could found in the active site of a serine protease?

Answer 16

critical serine residue

Question 17

point out the differences and similarities between the two phases the serine protease undergoes while explaining the process

Answer 17

phase one
1. hydrolysis: serine performs nucleophilic attack on the carbonyl-carbon (carbon of carbonyl :3) of the peptide bond, resulting in a scissile bond.
2. the nucleophilic attack is facilitated by Histidine 57 which acts as a general base to remove proton from serine (serine normally can’t release a proton). Histidine is a strong base because of the hydrogen bond with aspartate, a member of the triad. This results in a tetrahedral intermediate with a substrate that is covalently bonded to enzyme through the serine
3. tetrahedral intermediate has high energy and is unstable, therefore, it will decompose when Histidine 57 acts as an acid and gives back the proton to the nitrogen of the scissile peptide bond. This bond then dissociates, resulting in an acyl-enzyme intermediate which has 1/2 of the cleaved substrate.
4. remaining steps break the bond, release the remainder of the substrate, and regenerate the enzyme

phase two
1. hydrolysis: water attacks carbonyl-carbon of acyl-enzyme intermediate
2. the water attack is facilitated by Histidine 57 which acts a general base to remove proton from water. resulting deprotonated oxygen attacks the carbonyl-carbon. resulting in a tetrahedral intermediate (similar to phase one’s)
3. Histidine acts as acid and donates proton to serine’s active site to regenerate the original hydroxyl form and to release remainder of product

Question 18

in the process of the serine protease, what is the transition state?

Answer 18

tetrahedral intermediate

Question 19

in the process of the serine protease, what does the decomposition of the tetrahedral intermediate result in? (for both phases)

Answer 19

phase 1: formation of the acyl-enzyme intermediate and release of carboxyl-terminal half of the substrate

phase 2: release of amino terminal half of substrate and regeneration of original enzyme

Question 20

in regards to transition state, what is a major feature of enzymes?

Answer 20

enzymes can stabilize transition states

Question 21

describe the oxyanion and its process in regards to the tetrahedral intermediate

Answer 21

the tetrahedral intermediate contains an unstable oxyanion on the carbon

for the intermediate to be formed, the oxyanion on the carbon must be stabilized by an enzyme

when the tetrahedral intermediate forms, the oxyanion enters the oxyanion hole and then forms hydrogen bonds between the Histidine and Aspartate side chains which shorten - stabilizing it

Question 22

describe the specificity pocket

Answer 22

the specificity pocket allows for cleaving after different residues. it binds to residues and substrate that the enzymes specifically recognizes. this binding positions the peptide (scissile) bond optimally for cleaving by the active site residue.

Question 23

describe the specificity pocket for chymotrypsin and trypsin and elastase

Answer 23

chymotrypsin: must recognize bulky aromatic residues. serine at the base of pocket and glycine lining the pocket to make room for bulky side chains

trypsin: must recognize basic amino acid side chains. aspartate residue at base, allowing ionic interactions with the substrate

elastase: must recognize small aliphatic side chains. more bulky side chains linking the pocket

Question 24

what are serine proteases usually synthesized as? what is the purpose of this? give another example of this?

Answer 24

inactive precursors of an enzyme aka zymogens aka proenzymes

this allows enzyme activity to be controlled to prevent damage that may arise if left alone

another example of zymogens: in blood coagulation, all enzymes involved are circulating in the blood as zymogens. they only get activated when a wound occurs, at the site of the wound. otherwise, there would be uncontrolled coagulation through out the blood stream

Question 25

describe the activation process of trypsin and chymotrypsin

Answer 25

activated after secretion after being cleaved in 1 or 2 places by specific proteases

the trypsin is cleaved by enteropeptidase. the trypsin goes on to activate the chymotrypsinogen (zymogen form of chymotrypsin). chymotrypsin can also act on itself to make a few more clips but all of the cleaved forms are active – even the clipped structure retained through disulfide bonds

Question 26

at a fixed enzyme concentration, what is the relationship between the rate of the reaction and substrate concentration?

Answer 26

the rate of reaction will increase as the substrate concentration increases until a limit is approached.

as the enzyme becomes further saturated with substrate, further increases in substrate concentration does not increase the rate of reaction

Question 27

what is the michaelis-menten equation? when does this equation apply?

Answer 27

Vo = Vmax [S] / (Km + [S])

Vo - initial velocity

Vmax - maximal velocity (velocity at infinite substrate concentration at a given concentration of the enzyme)

S - substrate concentration

Km - michaelis constant. it equals the substrate concentration required to give a rate that’s half of Vmax

this equation only applies when the concentration of the initial complex is constant

Question 28

in the michaelis-menten equation, what is Vmax dependent on?

Answer 28

the Vmax of a reaction directly depends on the enzyme concentration (doubling the amount of enzyme will double the rate of product formation)

Question 29

describe Km and its relationships and relevance. why is it not an equilibrium constant?

Answer 29

Km is not dependent on the enzyme concentration. however, it is inversely related to the affinity of the enzyme for the substrate. (lower Km = higher affinity)

when the substrate concentration = Km, the rate is 1/2 the max rate (Vmax)

Km is not an equilibrium constant because although substrate binding is reversible, some of the bonded substrate turns over into the product

Question 30

how would one use the michaelis-menten equation?

Answer 30

plot equation using “double-reciprocal” plot or “lineweaver-burk” plot which
gives a straight line

double-reciprocal equation
1/V = (Km/Vmax)(1/[S]) + (1/Vmax)

this allows determination of Km & Vmax from velocity vs [Substrate] data on a graph y=mx+b

Question 31

what are the two classes of inhibitors?

Answer 31

irreversible: covalent bond with enzyme

reversible: non-covalent bond

Question 32

what type of inhibitor is penicillin?

Answer 32

irreversible

inhibits enzyme that contributes to cell wall synthesis - therefore, inhibiting organism’s growth

Question 33

describe reversible inhibitors

Answer 33

aka competitive inhibitors

binds directly in active site and blocks substrate binding

Question 34

describe the kinetic effect

Answer 34

increasing Km without affecting Vmax

this type of inhibition can be overcame with high substrate concentration

Question 35

how can the strong activity of enzymes be controlled?

Answer 35

control of enzyme abundance (through expression of its gene)

synthesis of enzymes as inactive precursors (zymogens) that must be activated

reversible modifications such as phosphorylation

allosteric regulation

Question 36

how do allosteric enzymes bind?

Answer 36

reversible binding of ligands at locations in the enzyme remote from the active site, that leads to alterations in enzyme activity.

similar to how protons bind to hemoglobin to promote T state

Question 37

describe allosteric regulation

Answer 37

process in which proteins transmit effects of binding at one site to another, often distal, functional site, allowing for regulation of activity - cooperativity!!

binding of regulatory molecule changes conformation of enzyme and affects its activity

usually, one enzyme in the pathway is regulated - the activity of this key enzyme would be repressed by the product of the pathway (negative feedback) & accelerated by precursors

allosteric regulators bind to enzyme or its regulatory subunits to promote T/R states

important feature of metabolic pathways

Question 38

what is required for cooperativity?

Answer 38

enzyme must have more than 1 subunit & more than 1 active site

Question 39

what example is given for allosteric regulation? describe it

Answer 39

Aspartate Transcarbamoylase (ATCase)

catalyzes in early steps of biosynthesis of nucleotide CTP

has catalytic subunits containing active sites and regulatory subunits which bind regulatory molecules (ATP & CTP)

it’s R state is more active

Michaelis-Menten doesn’t apply but Km & Vmax can still be measured

Question 40

when graphed, what does the chart of allosteric regulation yield?

Answer 40

its chart yields an S-curve (similar to hemoglobin) because as Aspartate concentration increases, more of the enzyme is shifted from the less active T-state to the more active R-state. when CTP binds, it slows down the enzyme, shifting curve to right. when ATP binds, speeds up enzyme.

Question 41

in allosteric regulation, what happens as the Aspartate concentration increases?

Answer 41

as Aspartate concentration increases, more of the enzyme is shifted from the less active T-state to the more active R-state. eventually, the enzyme becomes saturated with substrate. the cooperative behaviour allows rate of reaction to be controlled over wider ranges of substrate concentrations. if it wasn’t a cooperative enzyme, activity levels would shoot up to the max level even at low Aspartate concentrations

Question 42

why do metabolic pathways need to be controlled?

Answer 42

so right amount of synthesis occurs at the cell’s given conditions