Enzyme

Question 1

Importance of enzymes

Answer 1

Multiplicity of enzymes, their specificity and their susceptibility to regulation give cells the capacity to lower activation barriers selectively

Selectivity is crucial for the effective regulation of cellular processes

Question 2

What are enzymes?

Answer 2

Enzymes are biological catalyst that increases the rate of chemical reactions
- Mostly proteins but ribosomes can act as enzymes as well

Question 3

Characteristics of enzyme (Have 5)

Answer 3

• have extraordinary catalytic power, accelerating chemical reactions tremendously
• Show high degree of specificity for their substrate
• Function in aqueous solutions under very mild conditions of T and pH
• Have great capacity for regulation, including allosteric control, covalent modification, of enzymes, and variation of the amounts of enzymes synthesized
• Central to every biochemical process

Question 4

Describe the 4 types of enzyme specificity

Answer 4

1. Absolute specificity
   - Enzyme catalyzes only 1 reaction
   → Lactase catalyzes the degradation of lactose to glucose and galactose only
2. Group specificity
   - Enzyme will act only on the molecules that have specific functional groups like amino, phosphate, and methyl groups
   → Pepsin recognizes and cleaves aromatic amino acids like tyrosine, tryptophane and phenylamine
   → Trypsin can hydrolyze peptide bonds in which amino groups is contributed by any basic amino acids
3. Linkage specificity
   - Enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure
   → α-amylase can hydrolyze α-1,4-glycosidic bonds in starch and glycogen
   → Lipase can hydrolyze ester bonds between glycerol and fatty acids
   → Proteinase can hydrolyze peptide bonds between amino acids in the protein
4. Stereochemical specificity
   - Enzyme will act on particular steric or optical isomer
   → D-lactate dehydrogenase can catalyze the stereospecific interconversion of lactate and pyruvate (isomers)
   → β-glycosidase react only with β-glycosidic bonds which are present in cellulose
   → α-glycosidase reacts only with α-glycosidic bonds by starch and glycogen

Question 5

How do enzymes work?

Answer 5

Through the formation of enzyme-substrate complex in the active site of the enzyme, where catalysis takes place

The active site has specific shape complementary to the tertiary structure of the protein

Change in shape of protein or enzyme affects the shape of the active site and hence affects the function of the enzyme

Question 6

Specificity of active sites of enzyme

Answer 6

Surface of active site is lined with amino acid residues with substituent groups that bind the substrate and catalyze its chemical transformation

Only about 12 amino acid residues make up the active site, and only about 2 or 3 may be involved directly in the substrate binding and/or catalysis

ES complex is formed by binding of substrate to the active site through hydrogen bonding and other electrostatic interactions

Question 7

Which group in the amino acid is involved in actions of the enzyme and what are the functional groups that can play a catalytic role?

Answer 7

The side chain reactive groups are the ones involved in the action of the enzyme, except for hydrocarbon side chains

Functional groups include:
- Imidazole group of histidine
- Hydroxyl group of serine
- Carboxyl side chains of aspartate and glutamate
- Sulfhydryl group of cysteine
- Amino side chain of lysine
- Phenol group of tyrosine

Question 8

What does cofactor got to do with enzyme?

Answer 8

Cofactor binds to the active site of enzymes, covalently or non-covalently and is essential for the catalytic actions of enzymes that require cofactors

Question 9

How does metal ion act as a cofactor for enzymes

Answer 9

• Ionic interactions between enzyme-bound metal and a substrate can help orient the substrate for reaction or stabilize charged reaction transition states
• Form coordination compounds by behaving as Lewis acids (e- acceptor), while the groups they bind to act as Lewis base
  → have specific geometries which aid in positioning the groups involved in a reaction for optimum catalysis
• Mediates oxidation-reduction reactions by reversible changes in the metal ion’s oxidation state

Question 10

What is an enolase reaction?

Answer 10

The enolase reaction illustrates one type of metal ion catalysis and provides an additional example of general acid-base catalysis and transition state stabilization

Question 11

Describe a reaction that is catalyzed by enolase

Answer 11

Enolase converts 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate

The carboxyl group of 2-PGA is coordinated by 2 magnesium ions at the active site.

1. proton is abstracted by general based catalysis, forming an enolic intermediate stabilized by 2 Mg2+ ions
2. Elimination of -OH by general acid catalysis

Question 12

Briefly describe the 3 different enzyme-substrate models

Answer 12

1. Lock and key model
   - Interactions are rigid in nature
   - Both interaction interface are complementary in shape
   - Negligible conformational changes
2. Induced fit model
   - Conformational changes on binding
   - protein of varying degree of shape complementarity in unbound state to interact
   - Involves intrinsically disordered regions
3. Conformational-selection
   - Protein is dynamic in nature
   Interaction partners will explore different conformational states, but only particular states will be able to interact

Question 13

Describe the mechanisms of enzyme actions

Answer 13

• Enzymes function by lowering transition-state energies and energetic intermediates and by raising the ground-state energy
• Activation energy is the minimum energy required for the collision between reactants to give products
• Enzyme catalyzes reaction by providing an alternative pathway of lower activation energy, by providing a surface that complements the transition state in stereochemistry, polarity, and charge

*Binding of enzyme to the transition state is exergonic, and the energy release by the binding reduces the activation energy

Question 14

Does enzyme affect reaction rates or equilibria?

Answer 14

Enzymes affect reaction rate BUT does not affect reaction equilibria
- Equilibrium point of the process is unaffected
- Reaction only reach equilibrium faster when enzyme is present as the rate of reaction increases

Question 15

What is the transition state supposed to be, if not a chemical species?

Answer 15

Transition state is a fleeting molecular moment where bond breakage, bond formation, and charge development proceed to the precise point at which decay to either substrate or product is equally likely

Question 16

Concentration of substrate affects the rate of enzymatic reaction, then how do we measure rate of reaction?

Answer 16

Measure the initial rate (or initial velocity, V₀), when [S] is much greater than the concentration of enzyme, [E]
- At relatively low [S], V increases almost linearly with an increase in [S]
- At higher [S], V₀ increases by smaller and smaller amounts in response to increases in [S]
- When a point is reached beyond which increases in V₀ are vanishingly small as [S] increase (reach a plateau)

*Plateau-like region is close to the maximum velocity, Vmax
*The [S] required to reach half the Vmax is referred to as Km

Question 17

Saturation kinetics - what can be described at vmax?

Answer 17

1. virtually all enzymes are present as the E-S complex and free [E] is vanishingly small
2. Further increase in [S] will have no effect on rate

*This condition exist when [S] is sufficiently high that essentially all the free enzyme has been converted to the E-S form

After ES complex breaks down to yield the product, the enzyme is free to catalyze the reaction of another molecule

Question 18

When does the pre-steady state and the steady state occur in enzymatic reactions?

Answer 18

The initial period when enzyme is first mixed with a large excess of substrate is the pre-steady state, where the concentration of ES builds up

Pre-steady state only lasts for a few microseconds as the reaction is quick to achieve a steady state, where [ES] remains approximately constant over time

Question 19

What is the rate limiting step in enzymatic reactions?

Answer 19

The breakdown of the ES complex to product and free enzymes, P and E

Question 20

What is the rate equation of a one-substrate enzyme catalyzed reaction?

Answer 20

V₀ = (Vmax x [S])/(Km + [S])

V₀ = initial velocity rate
Vmax = maximum velocity
[S] = substrate concentration
Km = Michaelis constant, Km = (k₂ + k₋₁)/k₁

Question 21

k₂ refers to the rate of the ES -> P + E reaction
When k₂ is ___, k₂ &laquo_space;k₋₁ and Km = k₋₁/k₁, which is defined as the ___ constant, Kd, of the ES complex

This may not apply for most enzymes
Sometimes, k₂&raquo_space; k₋₁ and Km = k₂/k₁.

In other cases, k₂ and k₁ are comparable and Km remains a more ___ function of all 3 rate constants

Quantity Vmax also varies greatly from one enzyme to the next. If an enzyme reacts by the ___-step Michaelis-Menten mechanism, Vmax = ___, where k₂ is the rate limiting

Answer 21

k₂ refers to the rate of the ES -> P + E reaction
When k₂ is limiting, k₂ &laquo_space;k₋₁ and Km = k₋₁/k₁, which is defined as the dissociation constant, Kd, of the ES complex

This may not apply for most enzymes
Sometimes, k₂&raquo_space; k₋₁ and Km = k₂/k₁.

In other cases, k₂ and k₁ are comparable and Km remains a more complex function of all 3 rate constants

Quantity Vmax also varies greatly from one enzyme to the next. If an enzyme reacts by the 2-step Michaelis-Menten mechanism, Vmax = k₂[Et], where k₂ is the rate limiting

Question 22

If the product release EP → E + P, is the rate limiting reaction, the overall reaction can be described by:
E + S ⇌ (k₁/k₋₁) ES ⇌ (k₂/k₋₂) EP ⇌ (k₃) E + P

Most enzymes would be in the EP form at ___ and Vmax = ___

Answer 22

If the product release EP → E + P, is the rate limiting reaction, the overall reaction can be described by:
E + S ⇌ (k₁/k₋₁) ES ⇌ (k₂/k₋₂) EP ⇌ (k₃) E + P

Most enzymes would be in the EP form at saturation and Vmax = k₃[Et]

Question 23

What can be used to describe the limiting rate of any enzyme-catalyzed reaction at saturation?

Answer 23

A more general rate constant, Kcat, can be used. Kcat is the turnover number, which is equivalent to the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when enzyme is saturated with substrate

If reaction has several steps and one is clearly the rate-limiting, Kcat is equivalent to the rate constant for the limiting step

V₀ = (Kcat[Et][S]) / (Km + [S])

Question 24

How is Km and Vmax calculated from a graph?

Answer 24

Since the [s] against V₀ graph is exponential, 1/[S] against 1/V₀ is used instead to get a straight line of y= mx + b
The slope, m = Km/Vmax
Y-intercept, b = 1/Vmax

V₀ = Vmax[S] / Km + [S]
1/V₀ = Km + [S] / Vmax[S]
= Km/Vmax[S] + [S]/Vmax[S]
= Km/Vmax[S] + 1/Vmax

Question 25

Enzyme inhibitors are substances that interferes with the action of an enzyme and slows the rate of a reaction. Name the 4 different types of inhibitors.

Answer 25

1. Reversible inhibitor - Can bind to enzyme and subsequently be released, leaving the enzyme in its original condition 2. Irreversible inhibitor - Reacts with the enzyme to produce enzymatically inactive protein - Cannot regenerate the original enzyme 3. Allosteric inhibitor - Binds to a separate binding site outside the active center 4. Suicide inhibitor/mechanism-based inhibitor - Undergoes first few chemical steps of the normal enzymatic reaction - Instead of transformation into normal product, the inhibitor is converted to a highly reactive compound that combines irreversibly with the enzyme - Relatively inactive until it binds with the active site of enzyme - Reduces the side effects

Question 26

Describe the effect of competitive reversible inhibitor

Answer 26

Competitive inhibition - Occupies the active site and prevents the binding of substrate, by competing for the same site with substrate - No effect on Vmax, increased km - At low [S], inhibitor competes for the enzyme effectively - When [S] far exceeds [I], the probability of inhibitor binding to enzyme is minimized and the reaction exhibits normal Vmax

Question 27

Describe the effect of non competitive reversible inhibitor

Answer 27

Non-competitive inhibitor - Inhibitor does not resemble the conformational structure of the substrate, does not bind to active site, but on a separate site on the enzyme - Increasing [S] does not affect the % of active enzyme, % of inhibited enzyme remains the same - Reducing the amount of enzyme present reduces Vmax - Vmax is reduced, no effect on Km - Non-competitive inhibition effectively reduces the amount of enzyme by the same fixed amount at every [S] used - Km is constant for a given enzyme End-product/feedback inhibition is a non-competitive inhibitor → excess of the final product inhibits the enzyme and leads to a shut down of the series of reactions to prevent accumulation of intermediates in the pathway *Cytidine triphosphate binds to ATCase to prevent the reaction of its substrate, aspartate

Question 28

Describe the effect uncompetitive reversible inhibitor

Answer 28

- Uncompetitive inhibitors only bind to the ES complex at site distinct from the active site - Vmax decrease, Km decrease - The plot is parallel to the uninhibited reaction graph - Inhibitor-bound complex forms under high [S] and ES complex cannot release product while inhibitor is bound, reducing Vmax - Inhibitor bound complex effectively reduces the [ES complex], reducing Km - By le Chatelier's principle, a shift occurs to form more ES complex, resulting in less free enzymes and more enzyme in the form of ES or ESI (with the inhibitor) complexes

Question 29

Is enzyme activity dependent on temperature?

Answer 29

Yes. Temperature dependency is asymmetric, increasing temp increases thermal movement of molecules, leading to rate acceleration - At certain temp, enzymes become unstable and its activity is lost within a narrow temp difference due to denaturation *Optimal temp of enzymes in higher organisms rarely exceeds 50℃, but enzyme of thermophilic bacteria can go up to 100℃

Question 30

Is enzyme dependent on pH?

Answer 30

Yes. Enzymes are only active within a narrow range of pH (5-9) due to effects of pH on a combination of factors: 1. Binding of substrate to enzyme 2. Ionization states of the amino acid residue involved in catalytic activity of the enzyme 3. Ionization state of the enzyme 4. Variation of protein structure (at extreme pH) *Effect of removing a H+ from a.a. residue side chain - pH effect on enzymatic rate may reflect denaturation of the enzyme rather than protonation/deprotonation of specific catalytic residues → Type of a.a. residues involved can be identified by observing pH range in which enzyme undergoes changes - Buffers are used to keep enzymes in optimum or at least favourable [H+]

Question 31

How does phosphorylation control the activity of enzymes?

Answer 31

- The attachment of phosphoryl groups to specific a.a. residues of protein is catalyzed by protein kinases - Removal of phosphoryl groups is catalyzed by protein phosphatases - Addition of phosphoryl group introduces a bulky, charged group into moderately polar regions, while the oxygen in phosphoryl group can hydrogen bond with other groups in the protein

Question 32

Regulation of enzymes by phosphorylation is seen in glycogen phosphorylase of muscle and liver, which plays an important role in metabolism. Describe the process of glycogen phosphorylase activities.

Answer 32

Glycogen phosphorylase activity is subjected to allosteric control and covalent modification via phosphorylation It can exist in 2 forms: phosphorylated glycogen phosphorylase α and dephosphorylated glycogen phosphorylase β, which respond to different allosteric effectors, depending on the tissue type - α form is more active than β form

Question 33

What are other instances other than phosphorylation of glycogen phosphorylase where enzymes are controlled by phosphorylation?

Answer 33

Transport across membranes provides an important example: - Na+-K+ pump, where Na+ moves out and K+ moves in the cell - Only when ATP is hydrolyzed to ADP, enough energy is released to allow a number of energetically unfavourable reactions to take place

Question 34

How are enzymes classified and what are the 6 classes of enzymes?

Answer 34

Enzymes are classified by class, which can be narrowed down to their subclass and sub-subclass - Every enzyme is assigned 2 names and a 4-part classification number → e.g. aconitate hydratase, aconitase, EC 4.2.1.3 The 6 classes are: 1. Oxidoreductases - Dehydrogenases, oxidases, peroxidases, reductases, mono/dioxygenases 2. Transferases - C1-transferase, glycosyltransferases, aminotransferases, phosphotransferases 3. Hydrolases - Esterase, glycosidase, peptidase, amidase 4. Lyases - C-C/C-O/C-N/C-S-lyases 5. Isomerases - Epimerases, cis-trans isomerases, intramolecular transferases 6. Ligases - C-C/C-O/C-N/C-S-ligases

Question 35

NADH/NADPH are coenzymes to oxidoreductases, describe the function of NADH/NADPH.

Answer 35

NAD/NADP accepts a hydride ion from a reduced substrate NADH/NADPH donates a hydride ion to an oxidized substrate - Association between dehydrogenase and NAD/NADP is relatively loose and coenzyme readily diffuses from one enzyme to another, acting as water-soluble carrier of e- from a metabolite to another *Flavoproteins can participate in 1/2e- transfer, involving a grater diversity of reactions than NAD/NADP - Flavin nucleotide binds covalently to succinate dehydrogenase

Question 36

Describe the role of transferases

Answer 36

Transferases catalyzes transfer of a group from a molecule to another Kinases (a subclass to transferases): - Catalyze transfer of terminal phosphoryl group from ATP to accepter nucleophile Aminotransferase: - Transfer of α-amino group to the α-carbon atom of α-ketoglutarate, leaving behind α-keto acid analog of the a.a. - All transferase have the same prosthetic group and the same reaction mechanism → Prosthetic group: pyridoxal phosphate (PLP)

Question 37

Describe the role of hydrolases

Answer 37

Hydrolases catalyze hydrolysis reactions, which are responsible for enzymatic depolymerization of proteins, carbohydrates, and nucleic acids - Hydrolases catalyzes the hydrolytic cleavage of C-O, C-N, C-C, and P-N bonds - Active site geometry of hydrolases are all different, but they have the same catalytic method (hydrolysis) *Serine hydrolase enzyme family is one of the largest and most diverse enzyme classes

Question 38

Describe the role of ligases

Answer 38

Ligases can be entirely unrelated enzymes with the common feature of their ability to covalently link 2 substrates together, despite the reaction being unfavourable and requiring high energy DNA ligases: - Covalently links cloning vector and DNA to be clones - Joins Okazaki fragments together into a single DNA molecule Ubiquitin ligase: - Involves the covalent attachment of the low molecular weight of protein ubiquitin to its substrate proteins during the process of ubiquitination → E2 class of enzyme may form complex with E3 ubiquitin ligase to promote direct, or indirect transfer of ubiquitin

Question 39

Describe the role of lyases

Answer 39

Lyases are responsible for catalyzing addition and elimination reactions - Catalyzes the cleavage of C-C, C-O, C-N, and other bonds any means except hydrolysis or oxidation Isocitrate lyases: - Cleaves isocitrate, producing glyoxylate and succinate ATP citrate lyase: - Generates cytosolic acetyl-CoA and oxaloacetate by catalyzing de novo (new) fatty acid synthesis

Question 40

Describe the role is isomerases

Answer 40

Isomerases catalyze changes within a molecule, converting a molecule from 1 isomer to another Glucose-phosphate isomerase: - Catalyzes the conversion of glucose-6-phosphate to fructose-6-phosphate → Allows for formation of C=C, electrons from carbonyl forms O-H bonds with H donated by enzyme → Electron leaves the C=C bond to form C-H bond with proton donated by enzyme → Enzyme abstracts a proton, allowing the formation of C=O