1. ENZYMES

2. Topics covered are :- What are enzymes ? Cofactors General properties
classification
Nomenclature
Regulation of enzyme activity.
Factors affecting enzymatic
activity
Inhibitors

3. What are enzymes?
Enzymes are large biological molecules responsible for thousands of chemical interconversions that sustain life.
Enzymes are soluble ,colloidal organic catalyst ,specific in action , protein in nature.
They catalyze the hundreds of stepwise reactions that degrade nutrient molecules ,conserve and transform chemical energy from simple precursors.
Wilhelm Kühne first used the term enzymes.
For e.g. maltose is the substrate on which the enzyme maltase acts to form glucose .

4. Cofactor :-
Some enzymes require no chemical groups for activity ,others require some additional chemical component called as COFACTOR
They are non protein which active the enzyme when bound with it .
COFACTORS
ORGANIC
INORGANIC
PROSTHETIC
coenzymes

5. EXAMPLES:- INORGANIC- Cu⁺² cytochrome oxidase
Mg⁺² hexokinase, pyruvate kinase
ORGANIC-
Nicotinamide adenine dinucleotide Niacin
Flavin adenine dinucleotide riboflavin
PROSTHETIC-
NADH , NADPH

6. Enzymes are protein in nature ,when some cofactor attaches to it then enzyme is known as apoenzyme
An apoenzyme together with its cofactor is called a holoenzyme (this is the active form). (e.g. biotin in the enzyme pyruvate carboxylase).
Enzyme +cofactor active enzyme
(apoenzyme) (holozyme)

7. PROPERTIES OF ENZYMES :-
They are proteinaceous in nature.
They have more catalytic power as compared to inorganic catalyst
They have highly specific for their substrate
They accelerate the chemical reactions
Many inhibitors can affect the enzyme activity .
Enzyme have site which is lined with amino acids and this site is known as ACTIVE SITE.

8. Classification:-
The six major classes of enzyme with example are as follows :-
oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases

9. Oxidoreductases:- Transferases:- Defination-
Enzyme catalyzing oxido-reduction reactions .
E.g.-dehydrogenase
NAD NADH + H⁺
Transferases:-
Those enzyme transfering one group to other compound E.g. hexokinase
ATP + D-Hexose ADP + D-Hexose-6-phosphate .

10. Hydrolases:- Lyases :-
The enzymes which catalyze the hydrolytic cleavage of ester, ether, peptide, anhydride by addition of water.
E.g.- β-galactosidase
β-galactoside + H₂O alcohal +D-galactose
Lyases :-
Enzymes that catalyze removal of groups by leaving double bond . E.g.- aldolase
ketose -1-phosphate dihydroxy acetonephosphate+ an aldehyde.

11. Isomerases :- Ligases:-
Enzyme catalyzing inter conversion of optical ,geometrical or positional isomers.
E.g.- triose phosphate isomerase
D-glceraldehyde-3-phosphate Dihydroxy-acetone phosphate
Ligases:-
Enzymes catalyzing the linking together of two compounds . E.g.- succinate thiokinase
ATP+ acetly –CoA +Co₂ ADP+Pі +Succinyl-CoA

12. NOMENCLATURE :- E.C. no. – It stands for enzyme commission .
1) the first digit stands for class name
2) the second digit is for sub class
3) third and fourth digit stands for name of enzyme.
For e.g.-
ATP +D-Glucose ADP+D-glucose-6-phosphate
enzyme – Glucose phosphotransferase
Its E.C.no. is

13. The fist no. (2) denotes the class name i.e. transferase
Second no. (7) denotes the sub class phosphotransferase
third no. (1) denotes phosphotransferase with hydroxyl group as acceptor
Fourth no. (1) denotes D-glucose as the phosphoryl group acceptor .

14. Lock and key model :-
Enzymes are specific for particular substrate and it was suggested by the Nobel laureate organic chemist Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model.

15. How enzymes work ? This can be explained with the
help of reaction intermediate
diagram.

16. Important terms:-
Transition state – it is the state where maximum number of bonds breaks and form.
Activation energy –it is the minimum amount of energy required for conversion of reactants into products. It is denoted by Eact .

17. The starting point for forward or reverse reaction is ground state .
Free energy change denoted by ∆GO which plays important role.
In this coordinate diagram the free energy of products formation is less ,so formation of products is favored in this reaction i.e. the reaction is forward.

18. REGULATORY ENZYMES
These are the enzymes which increase or decrease the rate of whole of the pathway in response to certain signals.
They have multi subunit in which regulatory site and active site present mostly opposite to each other.
These are mainly of two types on the basis of their interactions:
Allosteric enzymes
Covalent enzymes

19. 1. Allosteric Enzymes:
These are the enzymes whose activity can be changed by molecules (effector molecule, modulator) other than substrate.

20. These are of two types, in response to the modulator:
These are reversible in nature.
These are of two types, in response to the modulator:
Negative Allosteric enzyme
Positive Allosteric enzyme

21. a) Negative Allosteric Enzymes:
These are the enzymes which are deactivated by the modulator by attaching with them.
These are reversible, therefore when the modulator is removed they are activated.
In them when modulator binds then the conformational change at active site doesn’t allow substrate to bind.

22. b) Positive Allosteric Enzymes:
These are the enzymes which are activated by the modulator by attaching with them.
These are reversible therefore when the modulator is removed they are deactivated.
For example, Phosphorylase-a is the enzyme which breakdown glycogen into glucose and Phosphorylase-b is inactive form.

23. Subunit interactions in an allosteric enzyme, and interactions with inhibitors and activators.

24. Allosteric enzymes does not follow Michaelis-Menten graph.

25. Allosteric enzymes show more rapid increase in activity, binding to the enzyme increases the activity much greater than that observed by Michaelis- Menten graph.

26. 2. Covalent Modifications:
These are the regulatory enzymes in which the modulators are bind with the covalent bonds at regulatory site.
It is reversible process .

27. For example, Phosphorylase-a is the enzyme which breakdown glycogen into glucose and Phosphorylase-b is inactive form.

28. ExampleS:- Phosphorylation :- (Tyr,Ser,Thr,His) ATP ADP O Enz Enz P O‾
Adenylylation :- (Tyr)
ATP PPі O
Enz Enz P O-CH₂ O adenine O⁻
H H H
OH OH

29. Uridylylation :-
UTP PPi O
Enz Enz P O CH₂ O uridine O‾ H
H H H
OH OH

30. TEMPERATURE- all enzymes have different optimum temperature
TEMPERATURE- all enzymes have different optimum temperature. With increase in temperature, collision frequency between enzyme and substrate increase. Therefore there is net increase in activity of enzymes. But after achieving optimum temperature i.e. temperature in which activity of enzyme is maximum, further increment in temperature activity decrease due to denaturation of protein. That’s why there is bell shaped graph.

31. pH- Enzyme have an optimum pH(or pH range) at which their activity is maximal, at higher or lower pH, activity decreases.
Any change in pH above or below the Optimum will quickly cause a decrease in the rate of reaction. If we provide acidic or alkaline condition, ionisation of enzymes occur, this results in charged changes in its active site and activity decrease.

32. CONCENTRATION OF SUBSTRATE- if initially increase the activity but after the formation of [ES] complex, graph becomes constant due to formation of [ES] complex which is denoted as steady state.
NOTE- steady state only occurs when there is excess of substrate.

33. CONCENTRATION OF PRODUCTS- as the product form, the collision frequency decrease because they interfere with the collision of substrate and enzyme.
Increase in product concentration , the enzyme activity decrease. This is known as FEEDBACK INHIBITION, occurs only in living system.

34. MICHAELIS-MENTEN EQUATION
MICHAELIS-MENTEN EQUATION -the rate equation for a one-substrate enzyme catalyzed reaction.

35. Vo = initial velocity
Vmax = max. velocity of enzyme which represent the interaction between enzyme and substrate
Km = concentration of substrate at which Vmax is half
[S]= substrate

36. LINEWEAVER BURT PLOT/ DOUBLE RECIPROCAL PLOT

37. TURN OVER NUMBER- it is number of substrate molecule converting into products per unit time by a single enzyme molecule. It is represented by K cat.
SPECIFICITY CONSTANT- Kcat/Km
As high as SPECIFICITY CONSTANT more efficient is an enzyme.

38. Inhibitors Of Enzymes
These are the certain unwanted compounds which affects the rate of reaction by reacting with the enzyme molecules.
On the basis of their nature, they are divided into two categories:-
Reversible Inhibitors
Irreversible Inhibitors

39. Reversible Inhibitors:
These inhibitors are reversible in nature.
On the basis of site of inhibition, these are of three types:
Competitive inhibitors
Uncompetitive Inhibitors
Mixed Inhibitors

40. Competitive Inhibitors:
These inhibitors competes with the substrate for the active site of an enzyme.
These inhibitors reduce the efficiency of the enzyme.

41. Competitive inhibition can be analyzed quantitatively by steady-state kinetics.
In the presence of a competitive inhibitor, the Michaelis - Menten equation is as follows:
The Line-Weaverburk plot is as follows:
From the graph, it is concluded that Km increases and Vmax remains unaffected.

42. ii. Uncompetitive Inhibitors:
These inhibitors binds at a site distinct from the substrate active site and, unlike a competitive inhibitor, binds only to ES complex.

43. In the presence of a uncompetitive inhibitor, the Michaelis - Menten equation is as follows:
The Line-Weaverburk plot is as follows:
An uncompetitive inhibitor lowers the measured Vmax and apparently Km also decreases.

44. iii. Mixed Inhibitors
These inhibitors also binds at a site distinct from the substrate active site, but it binds to either E or ES.
These inhibitors usually affects both Km and Vmax.

45. In the presence of a mixed inhibitor, the Michaelis - Menten equation is as follows:
From the graph it is concluded that Km increases and Vmax decreases.

46. II. Irreversible Inhibitors
These inhibitors bind covalently with or destroy a functional group on an enzyme that is essential for the enzyme’s activity, or those that form a particularly stable non-covalent association.
These are irreversible in nature.
Special class:
Suicide Inactivator:-
These compounds are relatively unreactive until they bind to the active site of a specific enzyme.