1. ENZYME- BIOLOGICAL CATALYST

2. Enzyme As Catalyst
All enzymes are proteins - with the exception of some RNAs that catalyze their own splicing all enzymes are proteins
In general, names end with suffix “ase”
- tyrosinase (tyrosine), celullase (cellulose), protease (protein), lipase (lipid)
Enzyme: a biological catalyst
enzymes can increase the rate of a reaction by a factor of up to 1020 over an uncatalyzed reaction

3. Catalysis: -
Catalyst:

4. Enzyme As Catalyst Enzymes are catalysts:
- increase the rate of a reaction
- not consumed by the reaction
- act repeatedly to increase the rate of reaction
- enzymes often “specific” – promote only 1 particular reaction, others catalyze a family of similar reactions
cellulase – cellulose as substrate
hexokinase – any 6 ring monosaccharide - fructose, glucose

5. General Properties of Enzyme
Higher reaction rates:
Milder reaction rates:
Greater reaction specificity:

6. Enzyme Catalysis
Active site - part of enzyme to which the substrate binds and the reaction takes place
Substrate – a reactant in an enzyme-catalyzed reaction
Product
Enzyme-substrate (ES) complex – the intermediate formed when the substrate is bind at the active site of an enzyme

7. Enzyme Catalysis
GENERAL FORMULA
E = enzyme
S = substrate
P = product

8. Enzyme catalysis reaction
Physically interact with their substrates to effect catalysis
E + S ES ES* EP E + P
Where:
- E = enzyme
- ES = enzyme/substrate complex
- ES* = enzyme/transition state complex
- EP = enzyme/product complex
- P = product

9. Enzyme catalysis reaction
Substrate bind to the enzyme’s active site
pocket in the enzyme
Catalytic site = active site = where reaction takes place

10. Enzyme catalysis reaction
E + S ES ES* EP E + P
1st step: enzyme binds to substrate molecule to form an enzyme – substrate complex
E + S ES
Enzyme

11. Enzyme catalysis reaction
E + S ES ES* EP E + P
2nd step: Formation of the transition state complex where the bound substance is neither product nor reactant
ES ES*, ES≠ES

12. Enzyme catalysis reaction
E + S ES ES* EP E + P
3rd step: Formation of the enzyme – product complex ES* EP

13. Enzyme catalysis reaction
E + S ES ES* EP E + P
4th step: Release of product EP E + P

14. Enzyme catalysis reaction
Enzyme can only work on one substrate molecule at
a time
Not change during the reaction
One product is release, enzyme is available to accept another substrate molecule

15. Enzyme Catalysis Rate of reaction = reaction velocity (V)
- the rate of enzyme reaction is measured by the rate of the appearance of products or the rate of disappearance of substrates.
- d[P]/dT or d[S]/dT
mol product/min or mol substrate/min
Enzyme activity?
1 unit (U) is the amount of enzyme that catalyses the reaction of 1 mol of substrate per minute under specified conditions.

16. Enzyme Catalysis
The rate of a reaction depends on its activation energy, DG°‡
an enzyme provides an alternative pathway with a lower activation energy
Activation energy – the energy required to start a reaction
Transition state – the intermediate stage in a reaction in which the old bonds break and new bonds are formed

17. How enzyme work? Transition state theory:
The enzyme (E) must approach the substrate (S), the substrate attach to the active site through noncovalent bond
Formed the high energy (unstable) ES complex
In ES complex, the covalent bond in substrate is in the process of breaking while the EP complex is forming.

18. Enzyme Catalysis - Example
Consider the reaction
catalase
No catalyst,
with added Fe3+ salt,
with added catalase

19. (b) – a/e lowered in the presence of an iron catalyst
(a) – a/e for the reaction in the absence of a catalyst
(b) – a/e lowered in the presence of an iron catalyst
(c) – energy diagram for the catalase-catalyzed breakdown of H2O2
(d) – energy diagram for the noncatalysed breakdown of H2O2 at elevated temperature
a/e – activation energy

20. Active site
Has specificity – can discriminate among possible substrate molecules
- others recognize a functional group (group specificity)
- only recognize one type of molecule (eg. D vs L isomer)
(absolute specificity)
Relatively small 3D region within the enzyme
- small cleft or crevice on a large protein
Substrates bind in active site by weak non-covalent interactions (Hydrogen bond, hydrophobic and ionic interaction)

21. Active site
The interactions hold the substrate in the proper orientation for most effective catalysis
The energy derived from these interactions – binding energy
Binding energy is used, in large part to lower the activation energy and stabilize the transition state complex (ES*)
Each non-covalent interaction provides energy to stabilize the transition state

22. Binding Models
Two models have been developed to describe formation of the enzyme-substrate complex
Lock-and-key model: substrate binds to that portion of the enzyme with a complementary shape
Induced fit model: binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit

23. Enzyme/substrate interaction
Lock and key model
- substrate (key) fits into a perfectly shaped space in the enzyme (lock)
- lots of similarities between the shape of the enzyme and the shape of the substrate
- highly stereospecific
- implies a very RIGID inflexible active site
- site is preformed and RIGID

24. Enzyme/substrate interaction
Induced fit model (hand in glove analogy)
- count the flexibility of proteins
- substrate fits into a general shape in the enzyme, causing the enzyme to change shape (conformation); close but not perfect fit of E + S
- change in protein configuration leads to a near perfect fit of substrate with enzyme
Figure 6.3, pg 148
Campbell & Farrell, Biochemistry, 6th Ed., 2009, Thomson Brooks/Cole

25. Key words for today You need to understand:
Factors effecting enzyme reaction rate
6 classes of enzymes
Cofactor, coenzymes, holoenzymes, apoenzymes
Allosteric enzyme, effectors (positive and negative), heterotropic and homotropic allosterism
Isoenzyme and multienzyme

26. Characteristics of enzyme reactions
What influence the enzyme reaction rate?
Substrate concentration
Temperature pH
Enzyme concentration
Inhibitor

27. Characteristics of enzyme reactions
Substrate Saturation:
[substrate] = substrate concentration

28. Characteristics of enzyme reactions
Temperature

29. Characteristics of enzyme reactions
Effects of Temperature:
All enzymes work within a range of temperature specific to the organism.
Increases in temperature lead to increases in reaction rates - is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates - due to the denaturating (alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding that stabilize the three dimensional structure of the enzyme.

30. Characteristics of enzyme reactionspH
pepsin in the stomach works best at a pH of 2 and trypsin at a pH of 8.

31. Characteristics of enzyme reactions
Effects of pH: Most enzymes are sensitive to pH and have specific ranges of activity.

32. Characteristics of enzyme reactions
Enzyme concentration

33. Characteristics of enzyme reactions
Inhibitor

34. Classification of Enzymes
Have 6 categories
Each enzyme has an official international name ending with –ase and a classification number
Number consists in 4 digits (referred to a class and subclass of reaction

35. Classification of Enzymes

36. Classification of Enzymes
Table 5.1, pg 136
Boyer, R., Concepts in Biochemistry, 3rd Ed., 2006, John Wiley &Sons

37. Enzyme Classes: Examples
Reaction 1
(oxidoreductase)
alcohol dehydrogenase 2
(transferase)
hexokinase
glucose + ATP  glucose-6-phosphate
+ ADP 3
(hydrolase)
chymotrypsin
polypeptide + H2O  peptides
More examples:Refer Table 5.2, pg 136 , Boyer, R., Concepts in Biochemistry, 3rd Ed., 2006, John Wiley &Sons

38. Enzyme Classes: Examples
Reaction 4
(lyase)
pyruvate
decarboxylase 5
(isomerases)
alanine
racemase
D-alanine L-alanine 6
(ligases)
carboxylase

39. Enzymes & cofactor
Enzymes require chemical entity in order to function properly (assists an enzyme in catalytic action)
Cofactor – nonprotein molecule that assist in an enzyme catalytic reaction
Coenzyme – smaller organic or organometallic molecule derived from vitamin, weakly bound to enzyme, temporarily associated with enzymes
Prosthetic group – coenzymes that are covalently or noncovalently tightly bound to enzyme and always present.

40. Enzymes & cofactor
Holoenzyme – an enzyme in its complete form including polypeptide(s) and cofactor
Apoenzyme – enzyme in its polypeptide form without any necessary prosthetic groups or cofactors

41. Allosteric Enzymes
Allosteric enzyme: an oligomer whose biological activity is affected by other substances binding to it
these substances change the enzyme’s activity by altering the conformation(s) of its 4°structure
Allosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an
allosteric inhibitor = negative effectors
allosteric activator = positive effectors

42. Allosteric enzyme in feedback Inhibition
Formation of product inhibits its continued production – feedback inhibition

43. Allosteric Enzymes (Cont’d)
The key to allosteric behavior is the existence of multiple forms for the 4°structure of the enzyme
allosteric effector: a substance that modifies the 4° structure of an allosteric enzyme
homotropic effects: allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase
heterotropic effects: allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP
ATCase = aspartate transcarbomylase
CTP = cytidine triphosphate

44. Allosteric enzymes
A change in conformational structure at one location of a multisubunit protein that causes a conformational change at another location on the protein
Effectors – i) serves as stimulants to enzyme (+ve effectors) = increase catalytic activity – ii) inhibitors (-ve effectors) to enzyme = reduce/inhibit catalytic activity
- Act by reversible, noncovalent binding to a site on the enzyme
Larger and more complex than nonallosteric enzyme
Have 2 or more subunits (oligomeric)
Allosteric enzymes have regulatory sites for binding of substrates and reaction (catalytic sites)

45. HOMOTROPIC ALLOSTERISM
Eg. Tetrameric allosteric enzyme composed of 4 identical subunits
Each subunit has a catalytic site where substrate/effector will bound and transformed to product
Once bound to active site, a message will transmitted via conformational changes to an active site on another subunit which makes it easier for a substrate molecule to bind and react at that site
This type (substrate and effector) are the same is called cooperative or homotropic

46. HETEROTROPIC ALLOSTERISM
A dimer with nonidentical subunits
Subunit α contain the active site – catalytic subunit
Subunit β contains the site for effector binding – regulatory subunit
Binding of a specific effector molecule to the regulatory site on the β subunit sends a signal via conformational changes to the catalytic site on subunit α
Substrate and effector different kinds of molecules - heterotropic

47. Isoenzymes
Enzymes that catalyze the same reaction (catalytically and structurally similar) but are encoded by different genes
Glycogen phosphorylase-synthesize in liver, brain and muscle-involves in degradation of glycogen
Isoenzymes = isoforms

48. Multienzymes
A group of noncovalently associated enzymes that catalyze 2 or more sequential steps in metabolic/biochemical pathway