CBS: Enzymes and pH/Buffers

Question 1

What are enzymes?

Answer 1

• Extremely efficient biological catalysts - decreases the transition state of a reaction, without altering the final equilibrium between reactants and products
• They should have a low affinity for the product so that it leaves the active site

Question 2

What is the difference between a transition state and an intermediate?

Answer 2

A transition state cannot be physically isolated (unlike an intermediate)

Question 3

Describe the way in which enzymes have specificity

Answer 3

• Enzymes will usually catalyse only one type of reaction e.g. alcohol dehydrogenase will only act on certain primary (not secondary) alcohols, oxidising them to aldehydes = group specificity
• Some enzymes are so specific that they will only act on one substrate = absolute specificity
• If a natural compound can exist in two stereoisomer forms (has a chiral centre), the enzyme concerned with its metabolism will usually only act on one isomer

Question 4

What is enzyme specificity determined by?

Answer 4

The presence of the active site - a region/cleft/groove of defined shape into which only the substrate of the correct shape and charge can fit/bind, allowing the reaction to take place

Question 5

What are the two consequences of enzyme specificity?

Answer 5

1) a group of enzymes present together in one compartment if a cell, working on one reaction with many steps - e.g. in the cytoplasm of muscle cells there is a complex and coordinate metabolic pathway in which the initial substrate D-glucose is converted through a sequence of specific enzyme catalysed reactions to the product (lactic acid)
2) a systematic classification scheme

Question 6

Describe the classification of enzymes

Answer 6

• Enzymes are divided into 6 main classes according to the type of reaction they catalyse
• The 6 classes are then further divided into sub groups according to their substrate/source
• Each enzyme is identified by its own individual 4 digit number

Question 7

What are the 6 main classes of enzymes and their type of reaction?

Answer 7

1) oxidoreductases - adds oxygen or remove 2H e.g. lactate dehydrogenase
2) transferases - transfer of functional groups
3) hydrolases - hydrolytic reactions
4) lyases - add groups to C=C bonds
5) isomerases - isomerisation reactions within the same substrate (transfer of functional groups within the same molecule)
6) ligases - form C-C or C-N bonds using ATP e.g. DNA ligases

Question 8

Describe enzyme structure

Answer 8

• Enzymes are proteins ∴ they are composed of one or more polypeptide chains folded into a complex and defined 3D shape
• The structure is stabilised by many weak bonds (H-bonds, electrostatic salt links and hydrophobic interactions)
• The active site contains functional groups that stabilise the transition state of the reaction

Question 9

What is the consequence of the weak bonds in enzyme structure?

Answer 9

• The weak bonds holding enzyme structure together are easily broken - e.g. heating the protein gives rise to a disorganised/tangled structure in which the enzyme no longer has any catalytic activity (inactive/denatured)
• This property makes enzymes very sensitive to changes in their environment e.g. increased pH protonates the enzymes ∴ there is lots of positive charge and the shape is lost (opposite with low pH, but the shape is still lost)

Question 10

What is the modified lock and key model for enzyme catalysis?

Answer 10

The substrate changes shape to fit into the active site which adapts but is intrinsically rigid

Question 11

What is the induced fit model for enzyme catalysis?

Answer 11

Like the modified lock and key, but the enzyme also has an intrinsic flexibility so both substrate and enzyme adapt to complement each other - e.g. carboxypeptidase A is open and then closes onto the substrate, isolating it for catalysis (relies on Zn2+ ion)

Question 12

What specific structure does the peptide bond catalysis by chymotrypsin (hydrolase) involve?

Answer 12

The catalytic triad (Ser, His and Asp) connected by H-bonds

Question 13

Explain the peptide bond catalysis by chymotrypsin

Answer 13

1) polypeptide substrate binds non-covalently with the side chains of the hydrophobic pocket of the enzyme via phenylalanine on the polypeptide to stabilise it
2) Ser is a very good nucleophile as its H is being pulled by His ∴ it binds to the substrate, forming a tetrahedral transition state and the H+ is transferred to His
3) H+ is transferred from His to the N on the C-N bond of the peptide (bond is broken), forming a free peptide and leaving behind an acyl-enzyme intermediate
4) a water molecule binds to His in place of the departed polypeptide (Ser bound to other half of substrate)
5) His makes the OH group of water a good nucleophile ∴ OH forms a tetrahedral transition state with the acyl intermediate and the H+ goes to His
6) the tetrahedral intermediate collapses, breaking the acyl bond, releasing the second peptide fragment with a COOH group (H+ returned back to Ser from His)

Question 14

Explain the effect of temperature on enzyme catalysed reactions

Answer 14

• Increased temperature increases the speed of the reaction and the ability to make more product
• The rate increases as collision between the enzyme and substrate is more likely due to increased kinetic energy
• At v high temperatures, the 3D shape is lost ∴ the shape of the active site and the ability to perform catalysis is lost
• Thermostability of enzymes is very important in organisms and business

Question 15

What is the effect of pH on enzyme catalysed reactions?

Answer 15

Different enzymes have different optimum pH based on the environment

Question 16

What do many enzymes rely on?

Answer 16

1) cofactors (metal) e.g. Mg2+ to make DNA redox inactive and Zn2+ used bc unreactive (redox inactive)
2) coenzymes e.g. NAD, FAD, ATP

Question 17

What are isoenzymes?

Answer 17

• Enzymes with different protein structures which catalyse the same reaction but are coded for by different genes
• They have distinct biochemical roles and are often found in different cellular compartments and in different amounts in different tissues
• e.g. lactate dehydrognase works differently in heart and muscle (diff isoenzymes)

Question 18

What is enzyme kinetics?

Answer 18

The study of the rate of an enzyme catalysed reaction, and how that rate varies with different [S], amounts of inhibitors, metal ions, cofactors and pH

Question 19

What is the reaction rate?

Answer 19

The increase in the amount of product formed/the decrease in the amount of substrate per unit time

Question 20

What are the axes on the Michaelis-Menten (M-M) plot?

Answer 20

X axis: [S] Y axis: reaction rate

Question 21

Describe the M-M plot

Answer 21

• For many enzymes, this follows a saturation hyperbolic function which tends to Vmax
• At low [S], the reaction rate is directly proportional to the [S] (first order kinetics)
• At high [S], the reaction rate is independent of the [S] (zero order kinetics)

Question 22

What is the M-M reaction model?

Answer 22

K1 K2
E + S <=> ES => E + P
K-1

Question 23

What are K1, K-1 and K2 in the M-M reaction model?

Answer 23

Rate constants

Question 24

Why is the ES => E + P reaction in the M-M reaction model not reversible?

Answer 24

Because the model is taken a very early stage of the reaction so there is v little product

Question 25

What are the 3 assumptions of the M-M reaction model?

Answer 25

1) [S]&raquo_space; [E] so that the amount of substrate bound by an enzymes at any one time is small
2) [ES} does not change with time (steady-state approximation) ∴ formation of ES = the breakdown of ES (to E+S/E+P)
3) initial reaction velocities are used ∴ [P] and the reaction of E+P => ES can be ignored

Question 26

What is the M-M equation?

Answer 26

V0 (initial reaction velocity) = (Vmax x [S])/Km + [S]

Question 27

What is the equation for the M-M constant (Km)?

Answer 27

Km = (K-1+K2)/K1

Question 28

What is Km?

Answer 28

The [S] at 1/2 Vmax

Question 29

Why does an enzyme with a lower Km have a higher affinity for its substrate (refer to Km equation)?

Answer 29

• If K2 ≈ 0, Km = K-1/K1 ∴ if Km is smaller, K1 > K-1 ∴ there is more formation of ES than breakdown of ES ∴ it has a higher affinity
• K-1/K1 = Kd (dissociation constant) ∴ lower Kd means less dissociation of ES

Question 30

When is Km useful?

Answer 30

To compare one enzyme’s affinity for different substrates but not to compare different enzymes on its own

Question 31

What is Kcat (turnover number) equivalent to?

Answer 31

The number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with substrate (higher = better)

Question 32

What is the best method to compare catalytic efficiency of enzymes?

Answer 32

Using the ratio Kcat/Km (higher = better)

Question 33

Describe the lineweaver-burke plot

Answer 33

• Rearranging the M-M equation to get it in the form of y=mx+c:
• 1/V0 = Km/Vmax x 1/[S] + 1/Vmax
• A plot of 1/V0 vs 1/[S] yields a straight line which can be used to determine Km and Vmax as well as the action of enzyme inhibitors
• 1/Vmax = y intercept and 1/Km = x intercept (by extrapolation)

Question 34

How can enzyme measurements be used in differential diagnosis of disease?

Answer 34

1) By investigating plasma levels of ‘escaped’ enzymes e.g. alanine aminotransferase and lactate dehydrogenase
- their levels in tissues may indicate what is happening
2) Laboratory estimations of metabolites in body fluids (blood, urine) e.g. glucose oxidase is used in Diastix (plasma) and Clinistix (urine) tests to estimate levels of glucose

Question 35

Describe the isoenzymes of lactate dehydrogenase

Answer 35

• Lactate dehydrogenase is composed of 4 monomers of which there are 2 types (heart or muscle)
• 5 different isoenzymes exist: H4, M4, H3M, H2M2, HM3
• The different isoenzymes have different kinetic properties that are tuned to the job the enzyme has to do where it is
• e.g. M4 in skeletal muscle has a high Kcat bc it has to get rid of the high amounts of lactate formed but not at such an urgent rate whereas H4 in the heart has a low Km to get rid the small amounts of dangerous lactate in the heart

Question 36

When is there a peak in LDH activity and why?

Answer 36

48h after infarction - during myocardial infarction, endothelial cells rupture, releasing their contents into the blood stream ∴ can diagnose it as there will be higher levels of H4 in the blood

Question 37

What are the 2 classes of enzyme inhibitors?

Answer 37

1) competitive (v similar shape) - blocks the enzyme active site by mimicking the substrate e.g. malonate inhibition of succinate dehydrogenase
2) non-competitive - binds somewhere else on the enzyme (not on the active site) but interferes in some other way with the catalytic mechanism (reversible or irreversible)

Question 38

What is the consequence of competitive inhibitors?

Answer 38

• They alter the apparent Km (not Vmax) as they decrease the affinity of the enzyme for the substrate
• Vmax is not altered as it corresponds to infinite [S]
• The slope of LWB plot increases but the y-intercept (1/Vmax) stays the same

Question 39

What is the consequence of non-competitive inhibitors?

Answer 39

• They do not affect Km as it binds to the active site ∴ the active site affinity for the substrate is the same
• However it does affect Vmax as even at infinite [S], the inhibitor’s effect will remain
• The slope decreases but the x-intercept (-1/Km) stays the same

Question 40

What are two clinical uses of enzyme inhibitors?

Answer 40

1) control of angiotensin production - a system which controls BP and relies on angiotensin converting enzyme (ACE) which converts angiotensin 1 to angiotensin 2, causing perpheral vasoconstriction (increasing BP)
2) control of acetylcholinesterase - breaks down ACh (ester bonds)

Question 41

Describe the enzyme inhibition of ACE

Answer 41

• An inhibitor of ACE can be used to control BP
• ACE is a metallo-enzyme (requires the presence of Zn centre)
• One inhibitor (captopril) binds to this Zn centre via the sulphur of its thiol group (H-S-Zn) - example of reversible inhibition

Question 42

Describe the enzyme inhibition of acetylcholinesterase

Answer 42

• The active site is very similar to chymotrypsin (with an identical catalytic triad)
• Example of reversible inhibition: neostigmine which binds to the active site, interacting with serine and forming an acyl-covalent compound - the active site then slowly regenerates
• Example of irreversible inhibition: diisopropyl fluorophosphate where the fluoride gets removed and phosphate binds directly to oxygen of serine covalently

Question 43

What are the 3 types of regulation of enzyme activity

Answer 43

1) allosteric binding sites - binding sites (effectors or inhibitors) that are different from active sites which provide a level of regulation
2) covalent modification by other enzymes e.g. phosphorylation by kinases, making enzymes less/more active
3) induction/repression of enzyme synthesis

Question 44

What is different about allosteric enzymes?

Answer 44

They show a sigmoid curve (instead of the M-M hyperbolic curve - like Hb dissociation curve

Question 45

Describe allosteric regulation

Answer 45

• Can involved positive effectors (have a positive effect on catalysis) or negative effectors (have a negative effect on catalysis) which bind to other sites on the enzyme
• Changes Vmax and Km
• Example of positive effectors: PEP and fructose 1,6 bis phosphate on pyruvate kinase
• Example of negative effectors: ATP and citrate on phosphofructokinase

Question 46

What are the types of covalent modification?

Answer 46

• Typically the addition/removal of phosphate from Ser, Thr, Tyr or His residues: 1) phosphorylation 2) dephosphorylation
  3) acetylation
  4) methylation

Question 47

Describe phosphorylation/dephosphorylation of enzymes

Answer 47

1) phosphorylation increases the activity of glycogen phosphorylase (degrades glycogen)
2) phosphorylation at several serine residues by glycogen synthase kinase 3 inactivates the enzyme - dephosphorylation occurs by phosphoprotein phosphatase
3) protein phosphorylation may lead to an increased or decreased activity or even catalysis of a different reaction

Question 48

Describe the induction/repression of enzyme synthesis

Answer 48

• High [blood glucose] leads to an increase in insulin production which increases the rate of synthesis of key enzymes involved in glucose metabolism: 1) glucokinase 2) pyruvate kinase 3) phosphofructokinase
• Controlled by a hormone/metabolite and can take hours/days