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BB10003 Biochemistry > Enzymes > Flashcards

Enzymes Flashcards

1
Q

What is an enzyme?

A
  • a biological catalyst
  • protein
  • operate under mild reaction conditions
  • specificity
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2
Q

What is a catalyst?

A
  • substance of material which accelerates a chemical reaction without being consumed
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3
Q

Chemical catalysis is in Hydrogenation of ethene on a metal surface

A

1) Surface chemo-adsorption of H2
2) Surface chemo-adsorption of ethene
3) electrophilic addition
4) rearrangement
5) De-adsorption of ethene product

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4
Q

Equation for chemical catalysis

A

Kcat / Kuncat

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5
Q

Internal asymmetrical active sites in enzymes

A
  • LYSOZYME
  • Reactions with negative ^G occur slowly/not at all due to activation energy
  • Energy input needed to convert reactions into unstable molecular forms called Transition State Species
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6
Q

transition state and activation energy determines…

A

rate of reaction

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7
Q

Effects of heat and pressure in chemical catalysis

A

Heat - speeds up reactions

Pressure - reduces entropy, increases FOSC

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8
Q

Effects of heat and pressure in Biological catalysis

A

Heat - increased –> denaturing of proteins

Pressure - increased –> ruptures cells

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9
Q

3 ways enzymes bind to substrates

A
  • lock and key
  • induced fit
  • transition state stabilisation model
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10
Q

Example of induced fit

A

hexokinase and glucose

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11
Q

In a transition state diagram what does ^G#uncat equal

A

Gibbs free energy of activation for the uncatalyzed reaction

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12
Q

Rate constant K depends exponentially on activation energy (transition state diagram)

A

K = Ae (^G#cat / RT)

- binding to enzyme stabilises transition state, reducing the transition state energy

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13
Q

4 factors that decrease Ae

A

1) enzyme holds reactants close together (FOSC)
2) enzyme produces microenvironment more suitable
3) enzyme puts strain on existing bonds –> break
4) active site of enzyme directly involved in reaction during transition states (diff pathway)

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14
Q

Stereo specificity

A
  • description of reaction path
  • enzymes can be highly specific in binding chiral substrates and catalysing
  • stereo-specificity due to enzyme active site geometry
  • termed ENANTIO-SELECTIVE
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15
Q

Example of Enantio-selective

A
  • YEAST ALCOHOL DEHYDROGENASE
  • ethanol is pro-chiral
  • ADH active site determines EtOH binding geometry
  • ADH transfers Pro-R hydrogen of EtOH to NAD+
  • CH3-CH2OH + NAD+ –> CH3-CH=O + NADH.H+
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16
Q

Geometric specificity

A
  • selective about chemical groups of substrate
  • few enzymes are absolutely specific for one substrate
  • some work on groups of related molecules e.g. Yeast ADH (primary and secondary alcohols)
  • some enzymes are permissive e.g. digestive enzymes (carboxypeptidases)
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17
Q

Coenzymes are…

A
  • metal ions or organic molecules
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18
Q

Cosubstrate is…

A
  • some coenzymes transiently associated with the enzyme
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19
Q

Prosthetic group is…

A
  • cofactors associated with the enzyme known as prosthetic groups
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20
Q

What is a holoenzyme?

A

catalytically active enzyme-cofactor complex

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21
Q

What is a Apoenzyme?

A

inactive protein (absence of cofactor)

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22
Q

Precursors and Vitamins

A
  • vitamins that’re precursors are H2O soluble

- Vit A and D (lipid soluble) aren’t components of coenzymes

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23
Q

Enzyme Assays

A
  • measure initial rate of product formation

- of substrate disappearance

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24
Q

Equation for enzyme assay

A

Rate v = dp/dt or -ds/dt (micromol/min)

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25
Q

Enzyme unit is

A
  • amount which gives 1 micromol prod/min
26
Q

Katal is

A
  • (SI unit) amount which gives 1mol/sec
27
Q

Direct Assays

A
  • way of detecting P or S directly dye to property of colour change or spectrophotometer, absorbing at certain wavelengths
  • all can be measured continuously
28
Q

Discontinuous Assays

A
  • reaction stopped at set times P and S measured

- possibly assay to measure P/S would inhibit the enzyme

29
Q

Example of discontinuous Assay

A
  • glucose measured with Hardings Test
  • Reduces CuSO4 to Cu2O when boiled in alkaline
  • green colour
  • measured in spectrophotometer
30
Q

Coupled assays

A
  • neither p or s can be measured
  • p can be consumed in another reaction and product of that can be measured
  • second enzyme must be in excess so that rate limiting step is one being measures
31
Q

example of coupled assays

A

alpha - glycerokinase

32
Q

Michealis Menton Kinetics, equation and assumptions

A

Equation: V = vmax [s] / Km + [S]

  • step ES to EP is irreversible
  • [ES] is in steady state
  • E>Km : V = Vmax
33
Q

What is Km?

A
  • Km of enzyme is the substrate conc at which the reaction occurs at half of the maximum rate
  • Km is diff between enzymes and for diff substrates
  • Km alters with temp and pH
  • higher the Km the high [S] needed to reach Vmax
34
Q

Catalytic Efficiency of enzymes

A

E + S ES E + P
Vmax = K2 [ET] = Kcat [ET]
- kinetic parameters provide a measure of its catalytic efficiency
- K2 is turnover number Kcat
- numver of reaction processes that each active site catalyses per unit time

35
Q

Lineweaver-Burk Plot

A

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

  • linearising it in double reciprocal form
  • 1/V0 = Km/Vmax (1/S) + 1/Vmax
  • y = mx + c
  • slope = Km/Vmax
  • Y = 1/vmax
  • X = -1/Km
36
Q

Effects of pH on enzyme activity

A
  • restricted to pH (typically 5-9)
  • regulates catalytic efficiency
  • substrate ionisation effects
  • protein structural changes
37
Q

Temperate effects on enzyme activity

A
  • increase in temp = increased flexibility in the backbone
  • increases activity
  • active site effects (reversible)
  • denaturation
38
Q

Types of enzyme regulation

A
  • positive or negative/activation or inhibition
  • covalent/non covalent interactions
  • covalent binding of regulatory molecule
  • non cov interaction of regulatory molecule
  • ionic, hydrophobic, van der waals
  • can be irreversible/reversible
  • post translational mods, protein cleavage, irreversible regulatory molecule binding
  • unfold quaternary structure
39
Q

Non Covalent regulation

A
  • reversible
  • highly specific - only target enzyme
  • 2 types - simple inhibitors, allosteric regulation
40
Q

Non covalent regulation binding to…

A

Enzyme = competitive
Enzyme + Enz Sub = non competitive
Enzyme Sub = uncompetitive

41
Q

Competitive inhibition

A
  • structure similar to substrate
  • occupies active site
  • inhibitor + substrate compete
  • binding of I + S mutually exclusive
  • inhibition reduced by increasing substrate conc
  • doesn’t affect vmax of enzyme
  • if [S] increases to infinity then all inhibitors displaced
  • at all [S], inhibitors will move eq to E from ES
  • Km will appear to increase to Kapp
42
Q

Equation for Competitive inhibition

A

KI (dissociation constant of EI) = [E] [I] / [EI]

43
Q

Non competitive inhibition

A
  • Molecule binds at remote site on enzyme in such a way that Kcat is affected
  • Km NOT affected
  • I + S bind at diff sites
  • Vmax reduced as catalytic rate reduced
  • point of intercept higher
44
Q

Equation for non competitive inhibition

A

V0 = Vmax [S] / ([S] + Km) (1 + [I] / KI)

45
Q

Uncompetitive inhibition

A
  • only when inhibitor binds with ES complex to make ESI
  • cannot yield products
  • not reversed by increase in [SUB]
  • found in reactions with two or more substrates
  • KM decreases
  • Increasing [I] diminishes Vmax + Km
  • but Km/Vmax remains constant
46
Q

Allosteric Inhibition

A
  • simple inhibition insufficient, highly sensitive
  • requires activation as well as inhibition
  • effector needs to be structurally unrelated to S
  • effector binds at another site on enzyme
  • neg (inhibitor) or positive (activator)
  • large amounts of effector needed
  • usually non competitive
  • effector binding alters protein conformation
  • activator improves substrate binding
  • inhib reduces this
47
Q

Allosteric proteins are

A
  • oligomeric
48
Q

Allosteric Inhibition with regards to V, [S] and [I]

A
  • V against [S] gives sigmoidal curve (doesn’t follow MM kinetics)
  • small change in [I] gives big inhibition (no change to Vmax)
  • small change in activator conc gives increase in V
49
Q

Covalent regulation

A
  • even allosteric regulation insufficient
  • where activity must be switched off reversibly
  • activation or inactivation
  • 2 types: - reversible: enzymatically interconvertible form
    Irreversible: enzyme activated enzymatically by cleavage
50
Q

Reversible covalent regulation

A
  • Very sensitive
  • Phosphorylation (pyruvate DH) suggested moves enzyme to higher energy state
  • adenylation (glutamine synthetase)
51
Q

Irreversible covalent regulation

A
  • enzyme potentially harmful, these enzymes synthesised as inactive precursors (zymogen)
  • e.g trypsin, if immediately active would digest host cells
52
Q

Oxidoreductases

A
  • oxidation / reduction reactions, transfer H and O atoms or electrons from substrate to another e.g. alcohol DH
53
Q

Transferases

A
  • transfer functional groups (methyl) from one compound to another e.g. hexokinase
54
Q

Hydrolases

A
  • catalyse hydrolytic cleavage of C-O, C-N, C-C and phosphoric anhydride bonds. e.g. carboxypeptidase
55
Q

Lyases

A
  • enzyme cleaving C-C, C-O, C-N, and others by elimination –> double bonds or rings or adding groups to double bonds e.g. pyruvate decarboxylase
56
Q

Isomerases

A
  • catalyse geometric or structural changes within one molecule e.g. maleate isomerase
57
Q

Ligases

A
  • catalyse joining of two molecules coupled with hydrolysis of a diphosphate bond e.g. pyruvate carboxylase
58
Q

Increasing temperature of an enzyme…

A
  • Increases the internal mobility of the enzyme tertiary structure
59
Q

The steady state assumption in enzyme kinetics assumes that….

A

[ES] remains constant over the time of measurement

60
Q

An enzyme catalyses a reaction by…

A

Decreasing the ΔGǂ of the reaction

BB10003 Biochemistry (5 decks)

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