1. Enzymes

2. Introduction
All chemical reactions in living organisms require enzymes to work for building , breaking down molecules or speed up the reactions.
Enzyme kinetics: the study of the rate at which an enzyme works.
Enzymes: are a biological catalyst. Usually a globular protein molecule produced by living organisms that can speed up a specific chemical reaction without itself being destroyed or changed in any way.
They are produced by the living organism and are usually present in only very small amounts in various cells.
proteins catalysts that speed up the rate of a chemical reaction by lowering the activation energy needed to convert the substrate to a product, without being used up in the process.

3. Enzymes properties Enzymes aren’t used up
Enzymes are not changed by the reaction
Re-used again for the same reaction with other molecules.
Most enzymes are proteins (tertiary and quaternary structures)
Act as catalyst to accelerates a reaction
Are specific for what they will catalyze

5. Enzyme Kinetics Equation

6. The Lock and Key Hypothesis
Enzyme may be used again
Enzyme-substrate complex E S P
Reaction coordinate
© 2007 Paul Billiet ODWS

7. Making réactions go faster?
Increasing the temperature make molecules move faster
Biological systems are very sensitive to temperature changes.
Enzymes can increase the rate of reactions without increasing the temperature.
They do this by lowering the activation energy.

8. Enzymes can increase the rate of reactions without increasing the temperature.
They do this by lowering the activation energy.

9. Factors affecting enzymes
Substrate concentration PH
Temperature
Inhibitors

10. Substrate concentration:
Reaction velocity
Substrate concentration
The increase in velocity is proportional to the substrate concentration
© 2007 Paul Billiet ODWS

11. Substrate concentration:
Reaction velocity
Substrate concentration
Vmax
Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. Vmax
© 2007 Paul Billiet ODWS

12. Michaelis and menten at low substrate concentrations, the enzyme is not saturated with the substrate and the reaction is not proceeding at maximum velocity whereas when the enzyme is saturated with substrate, maximum velocity is observed.

13. Michaelis-Menten Equation

14. PH Extreme pH levels will produce denaturation
Most human enzymes = PH 6-8
depends on it’s location in the body
pepsin (stomach) = pH 3
trypsin (small intestines) = pH 8
The structure of the enzyme is changed
The active site is distorted and the substrate molecules will no longer fit in it

15. Enzyme activity
Trypsin
Pepsin pH 1 3 5 7 9 11

16. Temperature Effect on rates of enzyme activity human enzymes
Optimum temperature
human enzymes
35°- 40°C (body temp = 37°C)
Raise temperature (boiling)
denature protein unfold lose shape
Lower temperature T°
molecules move slower
fewer collisions between enzyme & substrate

17. Temperture and enzymes Activity in human

18. Applications
Many are a lot lower, cold water fish will die at 30°C because their enzymes denature
A few bacteria have enzymes very high temperatures up to 100°C
Most enzymes however are fully denatured at 70°C
Thermophihlic bacteria

19. Inhibitors
Inhibitors are chemicals that reduce the rate of enzymatic reactions.
They are usually specific and they work at low concentrations.
They block the enzyme but they do not usually destroy it.

20. Reversible inhibitors:
Competitive
These compete with the substrate molecules for the active site.
The inhibitor’s action is proportional to its concentration.
Come over these problem by adding more substrate

21. Km : (Increased) The addition of a competitive inhibitor increases the observed Km for a given substrate.
Therefore, in the presence of a competitive inhibitor, more substrate is needed to achieve it.
Vmax: (not changed) Competitive inhibitors do not alter Vmax.
The effect of a competitive inhibitor is reversed by increasing [S]. high substrate concentration, the reaction velocity reaches the same Vmax as that observed in the absence of the inhibitor.
This is because at the higher concentration the active site will be saturated with substrate which means the inhibitor cannot bind

22. Irreversible inhibitors: Non-competitive: (allosteric effect)
These are not treated by the concentration of the substrate.
It inhibits by binding irreversibly to the enzyme but not at the active site.
Examples
Cyanide combines with the Iron in the enzymes cytochrome oxidase.

23. Km : (not changed) Non-competitive inhibitors do not interfere with the binding of substrate to enzyme.
Thus, the enzyme shows the same Km in the presence or absence of the non-competitive inhibitor.
Vmax: (decreased) Increasing the concentration of substrate does not overcome non-competitive inhibition.
Non-competitive inhibitors therefore decrease the Vmax of the reaction.
Non-competitive inhibitors therefore simply reduce the amount of active enzyme so they decrease Vmax, but have no effect on Km

25. competitive Inhibition: Km Increases; no change in Vmax
competitive Inhibition: Km Increases; no change in Vmax. Non-competitive inhibition: No Km change, but Vmax decreases

26. Glossary
Active site: The region of an enzyme molecule which binds the substrate and carries out the catalytic reaction
Enzyme: A biological catalyst. Usually a globular protein molecule produced by living organisms that can speed up a specific chemical reaction without itself being destroyed or changed in any way.
K m: (Michaelis constant) The substrate concentration at which an enzyme catalysed reaction proceeds at half the maximum velocity.
V max: (Maximum velocity): The maximum initial velocity of an enzyme catalysed reaction; determined by increasing the substrate [S] until a constant rate of product formation is achieved (i.e. saturating substrate levels).
A catalyst: is anything that speeds up a chemical reaction that is occurring slowly

27. Alkaline Phosphatase
Is a member of the phosphomonoestrases group. Which are highly specific and act on only one substrate, alkaline phosphatase has a broad substrate specificity and is named alkaline phosphatase because its PH optimum is usually around 9 but other broad spectrum phosphoesterases with PH optima less than 7 are termed acid phosphatases.

28. Determination of ALP in vitro
Alkaline phosphatase activity by following the rate of formation of products.
ALP catalyzes in alkaline medium the transfer of the phosphate group from 4 nitrophenylphosphate to 2 amino 2 methyle propanol, librating 4 nitrophenol
The catalytic concentration is determined from the rate of 4 nitrophenol formation, measured at 400nm.
The 4 nitrophenol is bright yellow but other reactants and products are colorless in aqueous solution.

29. The measurement of ALP activity in vitro is based on artificial substrate p-nitrophenylphosphate.
The intensity of yellow color in the reaction solution thus indicates the degree to which enzyme has acted upon substrate.(how much enzyme acted on substrate).

30. Study enzyme kinetics
Enzyme kinetics: the study of the rate at which an enzyme works.
To examine it, when the substrate available to the enzyme one would do the following:
set up the series of tubes containing graded concentrations of substrate
At time zero, a fixed amount of the enzyme preparation is added
Over the next few minutes, the concentration of product formed, is measured. If the product absorbs light, we can easily do this in a spectrophotometer
Early in the run, when the amount of substrate is in substantial excess to the amount of enzyme, the rate we observed is the initial of velocity Vi.

33. Thanks…