Enzymes

Question 1

Measure of enzyme activity

Answer 1

• enzyme can be assayed by exploiting its unique specificity for its substrates
• activity of a particular enzyme can be measured selectively by adding only its specific substrates
• applies even if enzyme present in complex mixture with other enzymes (eg crude tissue homogenate/ cell extract)
• requires only ability to monitor either fate of production formation (C or D) OR rate of substrate depletion (A or B)
• initial reaction rate (V0) - slope of initial linear portion of a graph of product formation (or substrate depletion) versus time. Rise over run of tangent.
• V0 is proportional to the amount of enzyme activity present
• continue and stopped enzymes available

Question 2

Continuous enzyme assays

Answer 2

Monitor the reaction process in real time
A. Simple (or direct) enzyme assays
- method of choice if any of the components of the reaction are directly measurable
- spectrophotometric light based approach common
- ideally exploit a chromogen that is directly involved in reaction, has high absorbance at a particular wavelength in visible / Uv spectrum, no other reaction component absorbed appreciably at same wavelength
B. Coupled (or linked) enzyme assays
- more complicated, but necessary if no component of reaction of interest can be measured directly
- involves coupling or linking the primary reaction via one of its products to another reaction which does include a compound that can be measured directly
- secondary reaction may be catalysed by another enzyme or may involve chemical step
- all components required for secondary reaction must be added in excess amounts, so that primary reaction remains the rate-limiting step in overall scheme

Question 3

Stopped enzyme assays

Answer 3

Required if neither of the previous continuous methods can be used
- reaction product not directly measurable, but conditions required to convert it o measurable compound are incompatible with enzyme function
- 2 step procedure:
A) enzyme incubation step
B) change chemical conditions to allow colour development during production detection step

Question 4

Measurement of enzyme activity must avoid:

Answer 4

Falsely low rates calluses by any “lag phase” (commonly seen early in coupled/ linked reactions)
Reduced rates caused by “substrate depletion” (always occur when a significant portion of the substrate has been consumed)

Question 5

Enzyme kinetics

Answer 5

Describe the behaviour of enzymes and the way enzymes respond to variations in environmental or experimental parameters.

• key factors is substrate concentration which changes during course of reaction
• so to simplify kinetics, we measure V0, the initial velocity of the reaction or the reaction rate at zero time.
• at very slow S, V0 increases almost linearly with S
• at moderate S levels, V0 increases by smaller and smaller amounts
• eventually at higher substrate levels, further increase in S cause essentially no further increase in V0 - approached asymptotically
• at this point, V0 approaches a kinetic parameter called Vmax, the maximal velocity of the reaction which occurs when the enzyme is fully saturated with substrate (infinite substrate concentration)
• michaelis constant Km or the affinity constant is the substrate concentration at which V0 is half maximal
• an enzymes Km is a measure of its affinity for its substrate. Inverse relationship between Km and affinity. Low Km= high affinity, enzyme becomes saturated with substrate readily at low S

V0 = (Vmax x S) \ (Km +S)

Question 6

Steady state kinetics

Answer 6

Early in raction before significant depletion of substrate
Concentration of E (free enzyme) and E-S (enzyme substrate complex) constant
Product formed at constant rate
Reaction rate is proportional to concentration of enzyme substrate complex [E-S]

Question 7

Problems trying to estimate Vmax and Km from V versus S plot

Answer 7

Although michaelis enzyme kinetics can be illustrated in form of a V0 versus S plot, this is not the ideal plot from which to determine the kinetic parameters Vmax and Km

• Vmax is therefore approached asymptotically, experimentally, plateau of maximal activity is never eactually reached, so Vmax can only be approximated from such a plot.
• Km is determined by calculating the S that induces half maximal reaction rates
• therefore any uncertainty in Vmax will necessarily lead to uncertainty of Km
• plus Interpolation on a curve is inherently inaccurate
• without prior knowledge of enzymes behaviour, experimental reaction rate data may not be collected at appropriate S values to enable determination of Vmax and/or Km

Question 8

Lineweaver- Burk plot problems

Answer 8

Inverse nature on both aces tends to emphasise least accurate points
Of a set reaction rates measured at evenly spaced S intervals, those measured at low S become spaced further apart, distant from the y axis - have greater bearing on position of line of best fit, but these are the least accurate data points
This factor can be overcome (at least partially) by choosing irregularly spaced S intervals

Question 9

Eadie-Hofstee plot advantages

Answer 9

Determination of Vmax and Km from this plot do not require extrapolation well beyond the range of data points.
Data on only one of the axes is subject to the problems associated with taking unversed of experimental values
The data points are spaced in such a way that the least accurate ones are not weighed or emphasised more than the accurate ones
Should be more relibe results for Vmax and Km

Question 10

Biological significance of Vmax

Answer 10

Vmax- maximal rate of enzyme catalysed reaction observed at infinite substrate concentration
Vmax provides no information about how fast reaction will proceed under physiological conditions
Useful:
- can compare Vmax values from multiple preparations of the same enzyme
- cannot compare Vmax values of different enzymes
- cannot compare raw Vmax values to be used to judge relative efficiencies or catalytic performances of different enzymes

Question 11

Biological significance of Km

Answer 11

Km is the substrate concentration that induces half maximal reaction rates and measure of an enzymes affinity for its substrate

• inverse/ reciprocal relationship: low Km means high enzyme affinity, high Km means low enzyme affinity for substrate
• the physiological significance of a Km value and its effect on enzyme activity can only be fully understood If the physiological concentration of the substrate is also considered.

Question 12

Gibbs free energy

Answer 12

If delta G> 0 (positive) reaction is energetically unfavourable, proceeds spontaneously from right to left. Gp > Gs

Gibbs free energy change deltaG= Gp - Gs

delta G has no measure of rate of reaction

Question 13

Activation energy

Answer 13

1. The transition state. S*, high energy form of S, intermediate structure between ground state of P, short lived transient
2. Activation energy delta Gact. Energy difference between ground state of S and the transition state
   Activation energy determines rate of reaction, energy hurdle, inverse relationship deltaGact low, reaction rate high
   Exponential non-linear relationship.
   Energy used for setting up appropriate alignments of reaction groups, formation of unstable short lived charged groups, the re-arrangement of bonds

Question 14

Active site

Answer 14

Lined with functional groups or other structural features which bind to the substrate
Functional groups can be provided by:
- r groups from particular amino acid residues in the enzymes sequence
- metal ions, coenzymes or cofactors associated with the enzyme
- energy required to lower delta Gact comes from multiple weak interactions between S and E
- every weak interaction releases a small amount of free energy
- sum of all these free energies is called binding energy delta Gb
- release of binding energy offsets activation energy

Question 15

Lock and key model

Answer 15

Active site is structurally complimentary to substrate. Fit perfectly together.

• explains ability of enzymes to bind to substrates
• explains unique specificity of enzymes for their substrates
• does not explain catalysis, whether is would cause an enhancement in reaction rate by making it easier to form the transition state somewhere between S and P
• does not explain reversible nature of enzyme

Question 16

Induced fit model

Answer 16

Shape of active site of enzyme is structurally complimentary to transition state of substrate, not to ground state of substrate.
Multiple weak interactions between S and E are optimised for transition state, not for ground state of substrate.
Formation of enzyme substrate complex makes it easier to form transition state.
Interactions between E and S help distort S towards the structure of the transition state- induce fit
Multiple weak interactions between E and S* release free energy- binding energy. This offsets activation energy of reaction.
- explains enzyme binding to substrate
- explains unique specificity of enzymes
- explains catalysis
- explains reversible nature

Question 17

Activation energy is essential

Answer 17

• apply to fwd and reverse reactions
• without these, complex polymeric macromolecules would rapidly break down back to their simpler lower energy monomers
• endless futile cycles of material from simple low energy precursors to complex macromolecules and back again
• complex molecular structures could never be maintained in the long term.

Question 18

Structural features the catalytic activity of enzymes depends on

Answer 18

• depends on integrity of native confirmation.. Each level of structure hierarchy requires that the previous level remains intact
• enzymes larger than their active sites
• active site must provide appropriate functional groups
• a small portion of enzymes structure is directly responsible for substrate binding and catalysis at active site
• must provide multiple weak interactions between E and S to help form S* so that delta G is reduced.

Question 19

pH activity profiles of enzymes

Answer 19

Usually bell shaped curve
Fairly symmetrical
- enzyme action involves amino acid side chains
- charge is pH dependent
- ionisable side chains may be: at active site, distant from active site or on the substrate itself with pH dependent charge
Enzymes have pH optimum
- at considerably lower or higher pH, ionisable groups on E are not either fully protonated or full deprotonated
- charge repulsion forces tend to liberalise
protein molecules
- at extreme pH proteins can be forced out of solution.
- pH optimum is usually close to pH of environment where enzyme normally functions
- buffering important in enzyme assays

Question 20

Temperature activity profiles of enzymes

Answer 20

• at low temp enzyme activity is very low due to low molecular vibrations
• added kinetic energy increases molecular vibrations, residue mobility. Greater probability of E and S being in correct orientation/ alignment so easier to overcome delta G activity and more likely to form transition state. Therefore enzyme activity increases at fairly steady rate.
• optimum temp- max enzyme activity, provides maximal product yield at given temp
• above optimum causes excessive molecular vibration and residue mobility. Weak interactions that normally stabilise protein structure are disrupted, thermal denaturation
• loss of structure causes loss of function
• temp control is usually essential for enzyme assays

Question 21

Thermal denaturation studies

Answer 21

• pre- incubating enzymes at different temperatures prior to assaying them at a standard temp.
• heating to a certain temp may selectively inactivate one enzyme activity while leaving another active
• thermal denaturation can also lead to precipitation of some proteins, so it can even be exploited to physically separate one protein from another.

Question 22

Chemical environment (various protein denatures)

Answer 22

Proteins usually arrange themselves with:- hydrophobic residues in core of protein (protected from water) and charged/ uncharged polar residues external (solvated/ H bonded with H20)
- chemical denaturants such as organic solvents, detergents, spouted such as urea guanidine hydrochloride
Denature proteins by interacting with normally internal hydrophobic residues

Question 23

Substrate specificity

Answer 23

The ability of an enzyme to discriminate between its normal substrate and another molecule.
Depends on:
- stereo chemical fit
- weak molecular interactions
- these two factors make up an induced fit. Ie distortion of structure of ground state of substrate towards its transition state
So the same forces that contribute to binding energy to reduce delta G also contribute to specificity

Question 24

General rules of substrate specificity

Answer 24

• if enzyme molecule has functional groups arranged to optimise weak interactions with a particular substrate in its transition state, it will be unable to interact to the same degree with any other molecule.
• an enzyme will exclude a molecule if it lacks a structural feature that is essential for binding in the substance
• a molecule with an extra functional group formation the enzyme has no pocket or binding site is also likely to be excluded from the enzyme
  Exception:
• binding of a molecule by enzyme rarely all or nothing process
• structural features that are non optimal may result in partial enzyme activity, not necessarily a total lack of activity
• some molecules structurally similar to substrate may be able to be acted on by the enzyme - bound by enzyme and acted in, just like substrate, depends on stringency of binding at the active site
• some molecules structurally similar to substrate may be able to bind at active site, but enzyme may not be able to act on them, act as inhibitors