Biology - Chapter 3 Enzymes

State 3 types of enzymes?

Catabolic: Breakdown substrate into 2 products
Anabolic: Combine substrates into 1 product
Metabolic: Speed up metabolic reactions

State 6 features of enzymes.

1. Bioloical catalysts
2. Specific to reaction
3. Unchanged at end of reaction
4. Effective in small amounts
5. High turnover no.
6. All globular proteins

Define turnover no.

The number of molecules of substrate that one molecule of an enzyme converts to product per second
(Max turnover no. = Vmax)

Explain how enzymes catalyse reactions.

Lower activation energy by providing an alternative pathway.

Define activation energy.

Energy needed for a chemical reaction to successfully form products

Define active site.

A site where substrates bind to & gives specificity as it is complementary in shape to substrate

State what the primary structure determines.

The shape of active site & the protein type

Contrast lock & key mechanism & induced fit mechanism.

Active site (X change shape VS flexible & moulds around S)
Degree of complementary (Fully VS Partially)
Fitness (Exact fit VS Better fit)

State & explain where enzymes operate.

Intracellular: Cell synthesise enzymes & retains them for internal use
Extracellular Cell synthesise enzymes & secrete them for external use (E.g. Lysozyme)

Explain te mode of action of an enzyme.

1. Enzymes & substrates move & collide randomly
2. Collisions in right orientation & enough energy form enzyme-substrate complex
3. Interactions of substrates with active site
4. Enzyme-product complex is briefly formed
5. Product leave active site while enzyme remains unchanged

Give the property of shape of active site of enzyme.

Specific that is complementary to substrate (X similar)

Describe how enzyme-substrate complex is formed.

S interact with R groups of catalytic amino acids at active site
1. Form temp bonds
2. Active site changes shape to mould around S (Ind. fit model)
3. S bind strongly to active site

Describe how the binding of S with active sites lower activation energy of reaction.

1. Bring S close tgt in right position
2. Put strain on them
-> Bonds can break / form easily
-> Allow easier transfer of charges / groups

Define rate of reaction.

Speed of conversion of S into product per unit time

Outline steps to measure ROR.

1. Measure rate of product formation / substrate disappearance
2. Plot a time course graph (time in x-axis; conc. in y)
3. Draw tangent & find gradient of curve (conc. / time) = ROR

Give how to calc. initial ROR.

Grad of curve at 0s

Define what a catalase is.

Enzyme found in tissues to break H2O2 down to oxygen & water.

State where catalase can be obtained from.

Potato / Liver / yeast extract

Give reaction for decomposition of H2O2.

2H2O2 (aq) -> (catalase) O2 (g) + 2H2O (l)

Outline how to measure ROR of catalse.

1. Measure rate of O2 released
2. Plot a curve of O2 produced against time
3. Draw tangent & find gradient of curve = ROR

State the general trend of ROR.

Decreases as reaction progresses

Give the hydrolysis of starch.

Starch -> (Amylase) Maltose

Outline how to measure ROR of amylase.

1. Measure rate of starch disappear by adding drop of I soln. at known time intervals
2. Use colorimeter to measure colour absorbance
2. Plot a curve of starch remaining against time (Need to know init. conc. of starch)
3. Draw tangent & find gradient of curve = ROR

State why is it not ideal to mix I, starch, and amylase in same tube.

I interferes with ROR by slowing it down

Describe how a colorimeter works.

Measures light absorbance through a cuvette (with sample in) in arbitrary units relative to the absorbance to control (distilled water)

Explain how [S] affects ROR. (5 pts)

At low [S],
1. Fewer collisions btwn S & E
2. Less S binds with active site
3. Not all active sites occupied
4. Few ESCs formed -> Low ROR
5. [S] is limiting factor

At high [S],
1. Rate inc. to plateau, Vmax is reached
2. All active sites saturated
3. Max no. of ESCs formed
4. [E] becomes limiting factor

State the trend of [P], [E], [S], [ES] in a reaction.

[P] inc. then plateaus at same level [S] started with

[E] constant as X use up

[S] dec. then plateaus

[ES] inc. from 0, then dec.

State 6 factors of enzymatic reactions.

1. [S]
2. [E]
3. Temp
4. pH
5. Inhibitors
6. SA

Define Vmax

Max rate of enzymatic reaction

Define Km

The [S] at which enzyme works at half its maximum rate (1/2 active sites are occupied by S)

Define affinity

The degree of attraction between molecules

Explain relationship between Km and affinity.

Inverse relationship
High affinity = great fit btwn active site & S
Takes less energy to successfully collide & form ESC
Less [S] needed to reach 1/2 Vmax -> Lower Km

State the axes of a double reciprocal graph.

1/v = y-axis
1/[S] = x-axis

State Vmax & Km on a double reciprocal graph.

Y-intercept = 1/Vmax
x-intercept = -1/Km

Explain how [E] affects ROR. (5 pts)

As [E] inc,
1. More enzymes present
2. More active sites available for S to bind
3. Inc. in frequency of collision
4. More ESCs are formed
5. Rate inc. as [E] inc.

At high [E],
1. [S] becomes limiting factor
2. Max no. of ESCs formed
3. ROR levels off

Explain why ROR on y-axis of ROR against [E] graph is initial ROR.

As reaction stretches, [S] dec, ROR dec.
Only diff. in ROR in initial stage of reaction caused by diff. in [E] -> Only use initial ROR in graph for reliability

Explain how temp affects ROR. (4;5)

As temp increases,
1. Increase in kinetic energy: E & S move faster
2. Increase in collision rate between S & E's active site
3. S bind to E's active site more often
4. More ESCs form

At optimum temp,
Max rate of enzyme reaction

At very high temp,
1. H bonds & ionic bonds holding enzyme start to break
2. Active site changes shape -> denatured
3. Substate cannot bind to active site
4. Fewer ESCs form
5. Reaction slows down drastically & stops - Irreversible process

Explain how pH affects ROR. (5 pts)

At low / high pH,
1. H+ ions interact with R groups of amino acids in enzymes
2. Disrupt ionic bonds & H bonds
3. Changes active site shape (denatured)
4. S canot bind to active site
5. Less ESCs form

At optimum pH,
Max rate of enzyme reaction
Suitable amount of H+ ions to interact with R groups of amino acids while keeping its shape
Not as irreversible as temperature

Define pH

Measure of conc. of H+ ions in soln.

Define inhibitors.

Molecules which can reduce rate of an enzyme catalysed reaction

State 2 places where inhibitors can bind & the type of inhibition.

Active site (Competitive, reversible inhibition)
Allosteric site (Non-competitive inibition)

State the properties of inhibitors in competitive, reversible inhibition. (3)

1. Similar to substrate's shape
2. Binds to active site
3. Non-permanent reversible binding -> Enzyme X damaged

Contrast the limiting factor of competitive, reversible inhibition.

Low [S]:
[I] > [S] -> Less freq. of collisions with S ->
Less ESCs formed -> Reduce ROR -> E's func. inhibited
High [S]:
[S] > [I] -> S outcompetes I & higher freq. of collision -> More S binded & ESCs formed -> E's func. unaffected

Compare & contrast Vmax & Km & ROR of ROR VS [S] curve for competitive, reversible inhibition.

Same Vmax as [S] outcompetes [I] so much I is insig.
S with I higher Km: Need more [S] to outcompete [I] to occupy half active sites with S
S with I lower ROR except when graph plateaus where its unaffected

State the properties of inhibitors in non-competitve, irreversible inhibition. (3)

1. Binds to allosteric site of E
2. Disrupt H bonds & hydrophobic interactions
3. 3D shape of enzyme affected due to distortion ripples -> Changes active site shape & no longer complementary

Compare & contrast Vmax & Km & ROR of ROR VS [S] curve for non-competitive, irreversible inhibition.

Lower ROR & Vmax as enzymes bound by inhibitors are destroyed -> Lower ROR as less available E

Same Km as E has same affinity for S -> E bound by inhibitors still attract S to active site & some S attracted to non-functioning E -> Same [S] needed to reach 1/2 Vmax

Explain the purpose of inhibitors in non-competitive, reversible inhibition.

Maintain homeostasis for metabolic reactions in a chain of reations by controlling amount of products formed

Explain how end-products inhibit reaction to control amount of products formed.

High amount of End product:
Binds to allosteric site on another enzyme catalysing upstream reaction in same metabolic chain
Inhibits reaction -> Dec. in amount of end products

Low amount of end product:
Inhibitor is temporary & can lose its attachment to be used elsewhere
Less enzymes inhibted so ROR inc.
More upstream products -> Inc. in amount of end products

Explain what is meant by immobilised enzymes.

Enzymes attached to inert, insoluble material through adsorption, covalent bonding, encapsulation, cross-linking

Outline how to encapsulate enzymes in alginate beads.

1. Mix enzymes with sodium alginate
2. Add drop by drop into CaCl2 (react to form jelly beads)
3. Pack them in a column (e.g. syringe with plunge pulled out & put a mesh in bottom)

State why we use immobilised enzymes. (8)

1. Can reuse
2. Can recover
3. Longer shelf life
4. Less purification / downstream processing needed
5. Allows continuous production with same column
6. Reduces end product inhibition as products are separated from enzymes
7. Enzymes more tolerant to pH changes
8. Thermostable - Less likely to denature at high temp

Define shelf-life.

The time during which enzyme may be stored & remain functional

Define Cofactors

Metal ions that bind (often permanently) to active site (Include prosthetic groups)

Define Coenzymes

Organic molecules (often vitamins) that function in the same way as cofactors but are often bound temporarily

Define allosteric enzymes.

Enzymes that have an additional binding site for inhibitors or activators to switch from active to inactive or even more active structure