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What is an enzyme?

Highly selective protein catalyst
May require a cofactor


What is a catalyst?

A substance that accelerates a chemical reaction without being consumed in the process


What are the thermodynamic properties of a reaction related to?

Energy balance and equilibrium


What are kinetic properties of a reaction related to?

Speed (velocity, or rate) of the reaction


What does enzymes do?

- Change kinetic but not the thermodynamic characteristics of the reaction
- Help process of substrate -> product
- Increases speed of achieving equilibrium (increase rate by lowering activation energy: delta G)
- Increase everse reaction (S -> P and P -> S)


What is the equilibrium constant?

Ratio of product concentration to substrate concentration at equilibrium


What are examples of cofactors?

1) Metal: Full of electron therefore can induce chemical reaction happen more easily. To gain and lose electron very easily (e.g. Fe2+)
2) Coenzyme


Name some inorganic ions that serve as cofactors for enzyme

1) Cu2+
2) Fe3+ or Fe2+ -> cytochrome oxidase, catalase, peroxidase
3) K+ -> Pyruvate kinase
4) Mg2+ -> hexokinase, G-6-P, pyruvate kinase
5) Mn2+
6) Mo
7) Ni2+
8) Se
9) Zn2+ -> alcohol dehydrogenase


What are examples of Coenzymes and examples of some of the chemical group transferred?

1) NAD => Hydride ion
2) NADP - nicotinamide adenine dinucleotide phosphate)
3) FAD - flavin adenine dinucleotide=> Electrons
4) FMN - Flavin mononucleotide


What does FAD do?

Gain or accept hydride ions and electrons


How are enzymes normally most commonly named?

After their substrate and their reaction type, with the suffix "-ase" at the end.
e.g. glucose oxidase


What are the main classes of enzyme classification?

1) Oxidoreductases
2) Transferases
3) Hydrolases
4) Lyases
5) Isomerases
6) Ligases


What type of reaction does oxidoreductases catalyse?

Catalyse oxidation-reduction reactions: electron transfer, hydrogen transfers, and reactions involving molecular oxygen.
- Transfer of electrons (e.g. hydride ions)


What does dehydrogenases transfer?

Transfer hydrogen between a substrate and a coenzyme (e.g. NAD, NADP, FAD, FMN)


Name examples of oxidoreductases

Dehydrogenases, oxygenase, peroxidase


What does oxygenase use?

Use molecular oxygen as a substrate.
Dioxygenase: incorporate both oxygen atom of O2 into their substrate (mono uses one)


What does peroxidase do?

Use hydrogen peroxide or an organic peroxide as one of their substate. Catalase is technically a peroxidase


What type of reactions does transferase catalyse)

Group transfer reactions


What is an example of transferase? What does it do?

Kinases: Transfer phosphate from ATP to a second substrate (Named according to the substrate to which the phosphate is transferred)


What type of reaction does hydrolyses catalyse?

Hydrolysis reaction (transfer of functional groups to water)
- Cleave bonds by the addition of water
- e.g. digestive and lysosomal enzymes


What type of reaction does lyases catalyse?

Addition of groups to double bounds, or formation of double bonds by removal of groups


What type of reaction does isomerases catalyse?

Transfer of groups within molecules to yield isomeric forms
- interconvert positional, geometric or optical isomers


What type of reaction does ligases catalyse?

Formation of C - C, C - S, C - O and C - N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor.
- often called syntheses or carboxylases
e.g. DNA ligase


What do all enzymes have?

Active sites


What are active sites?

Where chemical reaction take place and the substrate will bind long enough for chemical reaction to take place
- electrostatic reactions (hydrophilic & hydrophobic, hydrogen bonds, etc. )
- in the enzyme substrate complex


Does enzyme change the equilibrium (Keq) ?

Not change by enzyme
Determined only by Delta G(reaction)


What is Delta G(reaction)

Ratio of the two energies


What is the transition state?

Substrate must pass through this before the product is formed


What does active sites contain?

Functional groups for substrate binding and catalysis


What is the lock-and-key model?

Substrate and active site bind to each other because their surfaces are complementary.


What is induced fit model?

Many cases, substrate binding induces a conformational change in the active site that leads to further enzyme substrate interactions and brings catalytically active groups to the substrate
- after binding he substrate to guide the substrate though the intermediate as well


What is the enzyme substrate specificity determined by?

Geometry of enzyme substrate binding


Can reactions with a negative delta G occur?

Yes it can but in reality, may do not occur because in both catalysed and uncatalysed reaction,substrate must pass through a transition state before product is formed


What is the rate limiting step in the overall reaction?

The formation of the transition state


What is the free energy of activation?

the free energy difference between substrate and transition state
- energy barrier that must be overcome by the kinetic energy of the reacting molecules as they collide with each other


Most chemical reactions are meta-stable what does that mean/

Thermodynamically unstable
kinetically stable


How does the enzyme work to increase the rate of reaction

The enzyme decreases the free energy of activation


Does equilibrium of the reaction change when enzymes are used?

No, rate changes
- Small amount of energy change gives you big amount of equilibrium change


What happens when Keq increases in value?

Delta G becomes more negative


How do enzymes work?

Lock and Key Model (enzyme: lock & substrate: key)


Name the mechanisms for enzymes to work

1) Entropy effect
2) Stabilisation of transition state
3) Acid-base catalysis
4) Covalent catalysis


Explain entropy effect?

The transition state can form only when the substrate of a two substrate reaction collide in the correct geometric orientation and with sufficient energy to bring them to the transition state.
The enzymes increase likelihood by binding the two substrates to its active site in close proximity and in the correct geometric orientation


Explain stabilisation of transition state?

The enzyme forms favourable interactions with the transition state of the reaction thereby reducing its free energy content and the free energy of activation


Explain general acid base catalysis

Catalysis requires ionisable groups on the enzymes that accept or donate protons during the reaction.
Enzyme can provide electron pair donors and acceptors. Because the ionisable groups on the enzyme must be in the correct protonation state, general acid base catalysis is the most important reason for the pH dependence of enzymatic reactions


Explain covalent catalysis

The enzyme forms a transient covalent bond with the substrate


What's an additional mechanism to allow for enzymes to work?

Metal ion catalysis e.g. Fe++ <=> Fe+++ + e-
Use electrons to stabilise +ve charged intermediate or use Fe+++ to remove e-


Since lysine and arginine general acid form is mostly positive, what does that do?

Make it hard to remove the H from the NH3


How do you measure enzyme kinetics?

Measure initial velocity at different [S]


What is the relationship between rate and [S]

S -> P (but also S


What is Km?

substrate concentration at which the reaction rate is half-maximal
substrate concentration at which the enzyme is half saturated with its substrate


What unit is Km measured in?

substrate unit (e.g. mol/litre)


What does plotting a V vs [S] graph do?

Determine how good the enzyme is at catalysing the reaction
- Measure:
1) Effects of inhibitors
2) Compare enzymes
3) Understand how particular enzymes work


What are the 2 important parameters of the V vs [S] graph?

- Vmax: Maximum reaction rate
- Km: [S} that gives half Vmax


What does the V vs [S] graph show?

Almost all enzyme molecules are present as E-S complex, and the reaction is no longer limited by substrate availability but by the amount and turnover number of the enzyme.


In Michaelis Menten kinetics, what are the few simple assumptions made about enzyme catalysis?

1) Reaction has only one substrate
2) Substrate is present at much higher molar concentration than the enzyme
3) Only the initial reaction rate is considered, at a time when product is virtually absent and the backward reaction is negligible
4) The course of the reaction is observed for only a very short time period, the changes in substrate and product concentrations that take place s the reaction proceeds are neglected.


What cause the smell of intoxicated person's breath?



Liver ADH (Alcohol dehydrogenase) has lower Km for methanol or alcohol?

Lower Km for Alcohol than methanol


What does formaldehyde and formic acid do?

Formaldehyde: Chemically reactive
Formic acid: Causes acidosis
- Methanol poisoning: blindness and death


Whats the difference between ethanol and methanol metabolites?

Ethanol metabolites are channeled smoothly into the major metabolic pathways where they are rapidly oxidise to CO2 and H2O
Methanol metabolites accumulate in the body


Why is alcohol dehydrogenase used when a person has methanol poisoning?

Alcohol dehydrogenase can metabolise ethanol as well and it has a higher affinity for alcohol. Therefore the formation of methanol metabolites is delayed because ethanol compete with methanol for the enzyme.


How many different alcohol dehydrogenase are there in humans?

3 different ADH
- One low Km
- missing in some individuals
- if there are two high Km, only react in high conc.


What is the Lineweaver- Burk Plot?

Double reciprocal plot, 1/V against 1/{S} becomes a straight line


Why is the Lineweaver-Burk Plot needed?

in the curve, when measuring V against a different [S], its hard to fit curve


What are enzyme inhibitors

Most drugs are enzyme inhibitors e.g. aspirin that are reversible or irreversible


What are the 3 classes of reversible enzyme inhibition?

1) Competitive
2) Noncompetitive
3) Uncompetitive


What does competitive inhibitors do?

- inhibitor competes with the substrate by binding noncovalently to the active site of the enzyme


What does competitive inhibitors not change?

Vmax because inhibitor binding is reversible and can be overcome by high concentration of substrate


What does competitive inhibitors change?

Increase Km because substrate binding to the enzyme is impaired, and the apparent binding affinity is decreased


How do you achieve max velocity in competitive inhibition?

Put in enough substrate


What is a noncompetitive inhibitor?

Inhibitor binds to the second binding site, binds to E or ES and changes the shape of the E-S complex
- effective at low substrate concentration


What does noncompetitive inhibition do?

- Effectively removes enzyme
- lowers Vmax
- Km unchanged


What does uncompetitive inhibitor do?

bind only to the enzyme substrate complex but not to the free enzyme
- work best at high substrate concentration


How do you investigate inhibition?

Use changes in Vmax and Km


What does uncompetitive inhibitors do?

reduce both Vmax and Km


What curve would not yield a straight line in the Lineweaver-Burk Plot?

sigmoidal relationship between substrate concentration and reaction rate is typical for an allosteric enzyme with more than one active site and positive cooperativity between the active site - multi subunit (sigmoidal kinetics)


What does positive allosteric effectors do?

Activate the enzyme


What does negative allosteric effectors do?

Inhibit the enzyme


Where does the regulatory molecules bind to?

Bind to sites other than the substrate binding site
binding is non covalent therefore reversible


What can allosteric effectors do?

Change both the enzyme affinity for substrate(Km) and its turnover number


What does not conform Michaelis Menten kinetics?

Allosteric enzymes


How are chemical reaction affected by temperature?

Chemical reactions are accelerated by increased temperature
- The greater the activation energy, the greater is its temperature dependence


Protein denaturation is dependent on what?

Time dependent


Why are enzymes affected by pH

protonation state of catalytically active groups in the enzyme depends on pH


What is the optimal pH level of enzyme activity?

Lipase: 8.0
Pepsin: 1.5 - 1.6


What is the optimal temperature of enzyme activity?

37.0 °C


What are the factors affecting enzyme activity?

1) pH
2) temperature
3) substrate concentration
4) enzyme concentration
5) concentration of a substance


What is feedback inhibition

most biosynethtic pathways are inhibited by high concentrations of their end product


What does ATP stand for?

Adenosine Triphosphate


What is ATP used for?

- Energy carrier -> becomes ADP and AMP
- Phosphate donor for phosphorylation
- Act as coenzyme


What does NAD stand for?

Nicotinamide Adenine Dinucleotide


What are the reaction of NAD and NADP

1) NAD+ + 2H <=> NADH + H+
2) NADP+ + 2H <=> NADPH + H+


How is ATP formed?

Exergonic reactions are used for the synthesis of the energy rich compound ATP


What does the chemical bond energy of ATP do?

Drives the endergonic process


What is ATP?

A ribonucleotide, one of the precursor for ribonucleic acid synthesis
does not contain a vitamin
whole molecule can be synthesised from precurosrs


What are ATP made of and the bonds within?

First phosphate linked to ribose by a phosphate ester bond, but the two bonds between the phosphates are energy rich phosphoanhydride bonds