Enzymes and Enzyme Kinetics

Question 1

What are the characteristics of enzymes?

Answer 1

1) Enzymes increase the rate(s) of a reaction; enzymes can accelerate rxn’s as much as 10¹⁶ over uncatalyzed rxn’s
2) Enzymes are not consumed / destroyed during a chemical rxn
3) Enzymes may be localized to specific areas
4) Enzymes do NOT change the energetics of a reaction. That is, they do not change the delta G of a reaction.
5) Enzymes are present at low concentrations
6) Enzymes increase the probablity of a rxn occuring and therefore increase the rate of a rxn
7) Enzyme activity can be regulated

Question 2

Describe the physical appearance of an enzyme.

Answer 2

Enzymes are large, multi-subunit proteins. Their structure is vital to maintaining the integrity of he active site of the enzyme.

Question 3

Describe the active site of the enzyme.

Answer 3

The structure of an enzyme may serve to channel the substrate to the active sites binding residues.

The active site may contain cofactors or coenzymes that are essential for catalytic action.

Question 4

What is a cofactor?

Answer 4

A cofactor is a non-protein molecule that binds to an enzyme and is required for its biological activity.

Question 5

What are some common cofactors?

Answer 5

Fe²⁺

Cu²⁺

K⁺

Se

Zn²⁺

Question 6

What is a coenzyme?

Answer 6

A nonprotein organic substance that usually contains a vitamin or mineral and combines with a specific protein, the apoenzyme, to form an active enzyme system.

Question 7

What is a prosthetic group?

Answer 7

The nonprotein component of a protein. For example, the heme group in hemoglobin.

The nonprotein portion of the overall protein is usually covalently bound to the protein.

Question 8

True or False: Enzymes are specific.

Answer 8

True. Enzymes display a high degree of specificity and selectivetity for their substrate.

Question 9

What controls the specficity of an enzyme?

Answer 9

The structure of an enzyme controls its specificity.

Question 10

How may one get a clue to what reaction an enzyme catalyzes?

Answer 10

The enzyme name gives clues as to the reactions that are catalyzed by a particular enzyme.

Question 11

What are hydrolases?

Answer 11

Hydrolases are enzymes that catalyze hydrolytic cleavage reactions.

Question 12

What are nucleases?

Answer 12

Nucleases are enzymes that break down nucleic acids.

Question 13

What are proteases?

Answer 13

Proteases are enzymes that break down proteins.

Question 14

What are synthases?

Answer 14

Synthases are enzymes that are used in anabolic reactions that polymerize two smaller molecules.

Question 15

What are polymerases?

Answer 15

Polymerases are enzymes that catalyze the polymerization of nucleic acids.

Question 16

What are kinases?

Answer 16

Kinases are enzymes that catalyze the addition of a phosphate group to a molecule.

Protein kinases add a PO₄ to a protein.

Question 17

What are phosphatases?

Answer 17

Phosphatases catalyze the removal of a phosphate group.

Question 18

What are ATPases?

Answer 18

ATPases are enzymes that hydrolyze ATP.

Question 19

What are the six major classes of enzymes and what do they catalyze?

Answer 19

1) Oxidoreductases - Oxidation & Reduction rxn’s
2) Transferases - Group transfer
3) Hydrolases - Hydrolysis rxn’s (Transfers functional groups to water)
4) Lyases - Addition or removal of groups to form double bonds
5) Isomerases - Isomerization (intramolecular group transfer)
6) Ligases - Ligation of two substrates at the expense of ATP hydrolysis

Question 20

What is a nucleophile?

Answer 20

Nucleophiles are molecules that have relatively rich pi bonds or lone pairs that act as electron sources in the making of new bonds.

Question 21

What is an electrophile?

Answer 21

Electrophiles are molecules with relatively electron poor atoms and they serve as acceptors (or sinks) for electrons from nucleophiles.

Question 22

What are examples of nucleophiles?

Answer 22

Question 23

What are some common enzymatic reactions?

Answer 23

1) Make a bond between a nucleophile and an electrophile.
2) Break a bond so that stable molecules/ions are created
3) Add protons - The molecule has a strongly basic functional group or there is a strong acid present
4) Take a proton away - The molecule has a strongly acidic proton (amino acids) or there is a strong base present.

Question 24

How do enzymes work?

Answer 24

Enzymes work by lowering the activation enegry (Kinetic barrier) that a molecule needs to “become” a product.

Before a substarte can become a product, it must have a certain minimum energy to pass through a transition state.

Question 25

What is the significance of the transition state in enzyme catalyzed reactions?

Answer 25

When bonds are made/broken, charged intermediates are often formed which have a higher energy than the reactants.

The transition state is even higher in energy and may resemble the charged intermediate.

The goal, therefore, is to stabilize the charges and hence lower the energy of the transition state.

Question 26

Picture of transition state in enzyme catalyzed reactions.

Answer 26

Question 27

What is the ‘Lock and Key’ hypothesis of enzyme function?

Answer 27

The enzyme is the lock and the substrate is the key.

Question 28

What is the ‘Induced Fit’ hypothesis?

Answer 28

When a substarte binds an enzyme, a conformational change occurs in the enzyme.

This is the model that is prevalent in most enzyme-substrate interactions.

Non-covalent bonds stabilize the interactions between the enzyme and substrate.

Question 29

True or False: Enzymes are most complementary to substrate in the transition state rather than the ground state.

Answer 29

True; The primary job of the enzyme is to maximize favorable interactions with the transition state, NOT the starting substrates.

Enzymes bind the transition state more tightly than it binds the original substrates or products.

Question 30

True or False: Stabilizing the transition state results in enhancing the rate of catalysis.

Answer 30

True: After bond breaking/making at the transition state, the products are weakly bound by the enzyme. This results in loss of the product.

As bonds are broken/made, changes in charge may occur.

The enzyme can bind the transition state more tightly than the substarte because of the stabilization provided by direct interactions between the enzyme and transition state.

Question 31

What provides evidence of the transition state theory?

Answer 31

The tight binding of transition state analogs is evidence of the transition state theory.

Transition state analogs are stable molecules that are chemically / structurally similar to the transition state substrate.

Question 32

How does an enzyme invoke catalysis and accelerate the rate of reaction?

Answer 32

1) Stabilize transition state - Via electrostatic stabilization
2) Destabilize enzyme-substrate complex
3) destabilize intermediates

Question 33

How exactly does the stabilization of the transition state occur in enzyme catalysis?

Answer 33

Enzymes stabilize the transition state by the use of acid / base catalysis.

The catalysis is done by either Hydronim (H₃O) or Hydroxide (OH^- ).

Removing a proton decreases the developing of a positive charge.

Question 34

What are some of the important amino acid groups that are involved in stabilizing the transition state?

Answer 34

Asp

Glu

His

Cys

Tyr

Lys

Question 35

True or False: Enzymes can use acid and base catalysis simultaneously.

Answer 35

True

Question 36

True or False: The pKa values of an amino acid can be different in an amino acid side chain at the active site of an enzyme from those in solution.

Answer 36

True

Question 37

How do metal ions influence enzyme catalysis?

Answer 37

Metal ions can act as electrophiles and stabilize/shield the increased electron density and negative charge.

Metals can also be a nucleophile at neutral pH.

Metals can oreint the substrate and stabilize the charge on the substrate.

Question 38

What is the relationship of covalent or nucleophilic catalysis in enzyme-substrate reactions?

Answer 38

There is a transient formation of an enzyme-substrate covalent bond.

The nucleophilic group on the enzyme donates a proton to the electrophilic group on the substrate.

The original nucleophile can ten interact with the intermediate in a nucleophilic substitution rxn

The nucleophile catalyst and the original nucleophile interact w/ a carbonyl carbon in a substitution rxn and form a tetrahedral oxyanion intermediate

Question 39

What makes a good covalent catalyst and what are some examples?

Answer 39

A good covalent catalyst must be highly nucleophilic and form good leaving groups.

Examples of these would be imidazole and thiols

Lys

His

Cys

TPP

Question 40

Picture of Imidazlole ring.

Answer 40

Question 41

Picture of a thiol group.

Answer 41

Question 42

What does enzyme catalysis and proximity, orientation and strain have to do with one another?

Answer 42

Enzymes bring substrates and catalytic groups together

Enzymes orient these groups for catalysis

Enzymes facilitate attaining the transition state

Uses intermolecular and intramolecular strain to achieve this; Intramolecualr strain is faster at achieving catalysis

Question 43

How does destabilization of the enzyme-substrate complex and catalysis go hand-in-hand?

Answer 43

When the enzyme and substrate come together, entropy is lost

This reduces the translational and rotationaly mobility of the substrate

Desolvation makes some groups in the enzyme substrate complex more reactive by

a) Substrate has lower dielectric constant environment
b) Exposes H₂O bonded groups for electrostatic catalysis
c) Raises energy of the enzyme-substrate complex and makes it nmore reactive

Question 44

How can destabilization of the enzyme-substrate complex by electrostatic charges increase the rate of reaction?

Answer 44

Electrostatic destabilization of a substrate may result in a rate increase

This occurs because of the juxtaposition of like charges in the active site

This leads to charge repulsion that may be relieved in the course of a reaction

Question 45

What are enzyme kinetics?

Answer 45

Enzyme kinetics deals with factors that affect the rate of enzyme catalyzed reactions.

Enzyme concentration

pH

Ionic strength

Temperature

Ligand concentration

Question 46

What is the Michalis-Menten equation?

Answer 46

Km is the concentration of substrate that leads to half-maximal velocity. Km = 1/2 Vmax

Vmax is the limiting velocity as substrate concentrations get very large. Vmax (and V) are expressed in units of product formed per time.

If you know the molar concentration of enzyme, you can divide the observed velocity by the concentration of enzyme sites in the assay, and express Vmax as units of moles of product formed per second per mole of enzyme sites.

Question 47

What is Km?

Answer 47

Km is expressed in units of concentration, usually in Molar units.

Km is the concentration of substrate that leads to half-maximal velocity. Km = 1/2 Vmax

Small / low Km values mean tight binding of the enzyme to substrate

Large / high Km means weak binding of the enzyme and substrate

Km is a measure of how well an enzyme binds to a substrate

Question 48

How does concentration and Km go hand in hand?

Answer 48

If an enzyme is present and the substrate concentration is below the enzymes km, most of the enzymes binding sites will be free

If an enzyme is present and the substrate concentration equals the enzymes km, most of the enzyme binding sites will be taken

If an enzyme is present and the substrate concentration is above the enzymes km, then all of the enzyme binding sites will be taken up

Question 49

What is Vmax?

Answer 49

V_max is the maximum velocity or rate at which the enzyme catalyzed a reaction. It happens when all enzyme active sites are saturated with substrate.

Since the maximum velocity is described to be directly proportional to enzyme concentration, it can therefore be used to estimate enzyme concentration.

Vmax is never achieved as it is a theoretical maximal rate

Vmax is constant

To reach Vmax, all enzyme must be tightly bound to substrate

Vmax is asymptotically approached as substrate is increased

Question 50

What is turnover?

Answer 50

Turnover is measured by Kcat

It is a measure of catalytic activity of the enzyme

It specifically measures the number of substrate molecules converted to product per enzyme molecule per unit time when ezyme is saturated with substrate.

If the Michiles-Menten model is true, then K2 = Kcat = Vmax / Et

Question 51

What is the catalytic efficiency of an enzyme?

Answer 51

This is Kcat / Km

It is an estimate of ‘how perfect’ an enzyme is

This is a second rate-order constant

Measures how an enzyme functions when substrate concentration is low

The upper limit for Kcat / Km (108 to 109 M¹ sec¹ ) is the diffusion limit or the rate at which enzyme and substrate diffuse together

Question 52

Picture of Lineweaver-Burke equation.

Answer 52

Question 53

What are some ways that enzyme activity can be regulated?

Answer 53

10 Amount of substrate

2) Gene regulation
3) Regulatory molecules / Modulator proteins
4) Competition for active site
5) Allosteric regulation
6) Feedback control (Positive and negative)
7) Covalent modification
8) Phosphorylation / dephosphorylation
9) Cleavage (zymogens)
10) Isozymes - Enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters (e.g. different K_M values), or different regulatory properties.

Question 54

What factors effect the availability of an enzyme?

Answer 54

Compartmentalization

Transcription / translation

Protein processing / protein degredation

Question 55

Graph showing the effects of inhibition on an enzyme.

Answer 55

Question 56

Graph showing Michelis-Menten kinetics.

Answer 56

Question 57

Picture showing allosteric enzyme regulation.

Answer 57

Question 58

Picture showing allosteric negative feedback inhibition of an enzyme.

Answer 58

Question 59

Picture of enzyme positive feedback.

Answer 59

Question 60

Picture showing different modes of inhibition of an enzyme.

Answer 60

Question 61

What are the classes of reversible enzyme inhibitors?

Answer 61

a) Competitive inhibitors - Bind only the enzyme. Compete with substrate for enzyme binding site. Resemble the structure of the substrate. Lead to changes in Km but NOT Vmax. If substrate concentration is high enough, this can be overcome. Function as drugs. (See slide about Malonate inhibition of Succinate)
b) Uncompetitive inhibitors - This type of inhibitor binds to the enzyme-substrate complex. Keeps catalysis from happening by not allowing product to form. Change Vmax but not Km.
c) Noncompetitive / Mixed inhibition - Inhibitor binds to either the enzyme and/ o r the enzyme-substrate complex. It may or may not change substrate binding affinity. This alters the binding pocket of the enzyme. This is hypothetical.

Question 62

Graph of Michelis-Menten Non-competitive inhibition.

Answer 62

Question 63

Graph of Mechelis-Menten competive and non-competitive inhibition.

Answer 63

Question 64

Graphs of competitive inhibitors.

Answer 64

Km changes; Vmax does not change.

Question 65

Graph of uncompetitive inhibition.

Answer 65

Km and Vmax both change.

Question 66

Different types of inhibition.

Answer 66

NOTE: In mixed inhibition Km and Vmax both change.

Question 67

How does enzyme inhibition relate to metabolism?

Answer 67

Many end products are inhibitors of key steps in catalysis.

Drugs can be designed to inhbit key steps in a metabolic pathway.

Irreversible / covalent inhibition is accomplished using certain compounds. (Inhibit transition state)

Question 68

What is allosteric enzyme regulation?

Answer 68

Allosteric regulators bind at a site on the enzyme other than the active site

Enzymes that are situated at key steps in a metabolic pathway are modulated by allosteric regulators

These regulators are usually made elsewhere in a pathway

They may regulate by positive or negative feedback

Kinetics of allostericly regulated enzymes are sigmoidal )S-shaped)

This type of regulation leads to changes in concentration of enzyme and therefore change Vmax but not Km

Question 69

Picture of chymotrypsin binding site.

Answer 69

Question 70

Chymotrypsin mechanism of catalysis.

Answer 70

Question 71

Describe the chemistry of the active site of an enzyme.

Answer 71

The active site acts as a flexible structure that binds substrate and initiates the conversion to the transition state

By stabilizing the transition state, the enzyme greatly increases the concentration of the reactive intermediate that can be converted to product

Question 72

What are factors that affect reaction velocity?

Answer 72

Substrate concentration

• Maximal velocity: The rate or velocity of a reaction (V) is the number of substrate molecules converted to product per unit time. The rate of an enzyme catalyzed rxn increases w/ substrate conc. until V_max is reached.
• Shape of enzyme kinetics curves: Most enzymes show Michaelis-Menten kinetics which plots initial rxn velocity V_o against substrate conc. The curve is usually hyperbolic. Allosteric enzymes, however, show sigmoidal binding kinetics.

Temperature

• Increase of velocity with temperature: The reaction velocity increases with temperature until a peak velocity is reached.
• Decrease of velocity with higher temperature: Eurther elevating temperature causes a decrease in rxn velocity because of temperature-induced denaturation of the enzyme.

pH

• Ionization of active site: The concentration of H⁺ affects rxn velocity. The enzyme and substrate must have specific chemical groups in either an ionized or unionized state to be able to react.
• Denaturation: Extreme pH’s can denature enzymes

Question 73

What does V_max mean?

Answer 73

V_max is the maximum velocity or rate at which the enzyme catalyzed a reaction. It happens when all enzyme active sites are saturated with substrate.

Question 74

What does K_m mean?

Answer 74

K_m is the substrate concentration that gives half maximal velocity of an enzymatic reaction.

Measure of the affinity of enzyme and substrate with the higher the KM, the lower the affinity.

Called also Michaelis constant; Michaelis-Menten constant.

Question 75

What are some characterisctics of K_m?

Answer 75

K_m does not vary with enzyme concentration

Small K_m leads to a high binding affinity for substrate because a low concentration of substrate is required to half saturate the enzyme, that is, reach a velocity of 1/2 V_max

Large K_m lead to low affinity for substrate because a high conc. of substrate is needed to reach 1/2 V_max

Question 76

What is the relationship of velocity to enzyme concentration?

Answer 76

The rate of reaction is directly proportional to the enzyme concentration at all substrate concentrations.

For example, if the enzyme conc. is halved, the initial rate of reaction (V_o ) as well as V_max are reduced to half that of the original.

Question 77

How does competitive inhibition effect V_max and K_m ?

Answer 77

The effect of a competitive inhibitor on V_max is reversed by incresing substrate conc.

Competitive inhibition increases the apparent K_m . This means that in the presence of a competitive inhibitor, more substrate is needed to achieve 1/2 V_max

Question 78

How do non-competitive inhibitors effect V_max and K_m ?

Answer 78

Non-competitive inhibitors decrease apparent V_max as it cannot be overcome by increasing substrate conc.

Non-competitive inhibitors do not interfere with the binding of substrate to enzyme. As such, they have no effect on K_m .

Question 79

What is an isozyme?

Answer 79

These are enzymes that catalyze the same reactions but do not have the same physical properties because of their different amino acid sequences.

Question 80

What are the traits of an allosteric enzyme?

Answer 80

a) Composed of multiple subunits
b) Catalyze the committed step
c) Bind substrate cooperatively
d) Show a sigmoid binding curve whne V_o is plotted against [S]

Question 81

What are the mechanisms of enzyme catalysis?

Answer 81

Substrate proximity and orientation

Acid / base catalysis

Question 82

Describe the reaction mechanism of chymotrypsin.

Answer 82

Chymotrypsin uses acid / base catalysis

There is a serine at the active site that acts as a base / proton acceptor during the hydrolysis of a peptide bond

Step I: The serine in the enzymes active site attacks the peptide bond to be cleaved

Step II: A covalent complex is formed between the enzyme and the portion of the substrate that is to be cleaved

Step III: The enzyme - substrate complex is hydrolyzed which releases the peptide