1. Enzymes Nearly all the reactions of the body are mediated by enzymes
Enzymes are protein catalysts that increase the rate of the reactions without being changed in the overall process
Catalysts for biological reactions
Lower the activation energy
Increase the rate of reaction
Activity lost if denatured
May be simple proteins
May contain cofactors such as metal ions or organic (vitamins)

2. Enzymes Nomenclature of enzymes Enzyme has two names
a. Short Recommended name
b. Systematic name
Recommended name
End in –ase,
Identifies a reacting substance
sucrase – reacts sucrose
lipase - reacts lipid
Describes function of enzyme
oxidase – catalyzes oxidation
hydrolase – catalyzes hydrolysis
lactate dehydrogenase, adenylate cyclase…
Common names of digestion enzymes still use which don't provide any hint as pepsin and trypsin

3. Systematic name
The international union of Biochemistry and Molecular Biology (IUBMB) developed a system for nomenclature in which enzymes are divided into 6 groups and sub classes. These names are unambiguous and informative but sometimes long and difficult to be of general use.
Class Reactions catalyzed
Oxidoreductoases oxidation-reduction
Transferases transfer group of atoms
Hydrolases hydrolysis
Lyases add/remove atoms to/from a double bond
Isomerases rearrange atoms
Ligases combine molecules using ATP

4. Enzyme Action: Lock and Key Model
An enzyme binds a substrate in a region called the active site
Active site is a special pocket or cleft in the enzyme molecule
The active site contains amino acids side chains that form a three dimensional surface complementary to the substrate
Only certain substrates can fit the active site
The active site binds to the substrate and form enzyme-substrate complex that will dissociate into the enzyme and product.
Amino acid R groups in the active site help substrate bind

5. Lock and Key
The active site of the unbound enzyme is complementary in shape to that of the substrate

6. Enzyme Action: Induced Fit Model
Enzyme structure flexible, not rigid
Enzyme and active site adjust shape to bind substrate
Increases range of substrate specificity
Shape changes also improve catalysis during reaction
The enzyme changes shape upon binding substrate
The active site has a shape complementary to that of the substrate only after the binding

7. Enzyme-Substrate Complex

8. Cofactors
Some enzymes associate with non-protein cofactor that is needed for enzymatic activity
These cofactor include metal ions (Zn, Fe) and organic molecules called coenzymes that often derivative of vitamins; NAD+, FAD, CoenzymeA..
Holoenzyme refers to the enzyme with its cofactor, Apoenzyme refers to the protein portion of the holoenzyme and it dose not show biological activity.
Prosthetic group is a tightly bound enzyme that dose not dissociate from the enzyme
Location of enzymes
Many enzymes are located into specific organelles in the cell serve to isolate the reaction substrate or product from each other and to provide a special environment for a reaction and to organize these reactions

9. Turnover number and catalytic efficiency
Enzyme-catalyzed reactions are highly efficient, proceeding from 103 – 108 times faster than unanalyzed reactions.
The number of substrate molecules converted into product per enzyme per second is called Turnover number, typically each enzyme molecule is capable of transforming of substrate molecules into product per second
Enzymes are highly specific, interacting with one or a few specific substrate
Enzymes could be enantiomers specific

10. How Enzymes work
The mechanism of action of enzymes can explained by two different modes
Energy changes during the enzyme-catalyzed reaction, enzyme provide an energetically favorable reaction pathway different from unanalyzed reaction
The active site chemically facilitates catalysis
Energy Changes during the reaction
- the reactant and the product are separated by energy gap or energy barrier that called free energy of activation and it equals to the energy difference between the energy of reactant and the energy of high-energy intermediate (Transition state)
A  T*  B
The peak of energy represents the Transition state in which the high-energy intermediate is formed during the conversion of reactant into product.
because of large activation energy the rate of unanalyzed reaction is slow

12. Rate of Reaction
-The substrate molecules should have sufficient energy to overcome energy barrier to be converted into products.
only few molecules have energy to pass the energy gap  rate of reaction is determined by number of molecules that is converted into the product.
the lower the free energy of activation, the more molecules have sufficient energy to pass over the transition state the faster the rate of the reaction

13. The enzymes don’t change the free energies of the reactants and products  don’t change the equilibrium of the reaction

16. Chemistry of the active site
The binding of a substrate to the binding site involves a complex molecular machine chemical reactions  facilitate the conversion of substrate to product.
Many factors responsible for enzyme catalytic efficiency of enzymes:
Transition state stabilization: the active site often act as a flexible molecular template that binds substrate  stabilize the transition state  increase the product production
Active site provide catalytic groups that enhance the probability that transition state forms, these groups participate in general acid base catalysis in which amino acids can accept or provide protons, in other enzymes catalysis may involve the transient formation of a covalent enzyme-substrate complex

17. Transition State

18. Factors Affecting Enzyme Action
Different enzymes show different response to changes in substrate concentration, temperature, and pH
Reaction Rate (velocity of a reaction):
Reaction rate is the number of substrate molecules converted to product per unit time and is usually expressed as µmoles product formed per minute
The rate of reaction (enzyme catalyzed) increases with substrate concentration until a maximal velocity (Vmax), the plateau reflects the saturation with a substrate of all available binding sites on the enzyme
substrate concentration
Maximum activity
Reaction Rate
Increasing substrate concentration increases the rate of reaction (enzyme concentration is constant)
Maximum activity reached when all of enzyme combines with substrate

19. substrate concentration
Maximum activity
Reaction Rate
Vmax
Vmax/2 Km
Most of enzymes show hyperbolic dependence of velocity on substrate concentration

20. Factors Affecting Enzyme Action: Temperature
Little activity at low temperature
Rate increases with temperature (the velocity increased with Tem until a peak due to the increased number of molecules having sufficient energy to pass over the energy barrier and form product)
Most active at optimum temperatures (usually 37°C in humans)
Activity lost with denaturation at high temperatures decrease the velocity
Optimum temperature
Temperature
Reaction Rate
High
Low

21. Factors Affecting Enzyme Action: pH
pH affect the ionization of the amino acids in the active site.
R groups of amino acids in the active site should have proper charge to bind with the substrate the ionization or unionization is affected by the pH
Maximum activity at optimum pH
Tertiary structure of enzyme is correct
Most lose activity in low or high pH, extremes of pH can also lead to denaturation of the enzyme because the structure of active protein depends on the ionic character of the amino acid pH
Reaction Rate
Optimum pH
Narrow range of activity, and the pH optimum varies for different enzymes

22. Each enzyme has its optimum pH

23. E + S ES E + P V [S] Rate v = km + [S] Michaelis-Menten Model k1 k2
Michalis and Menten proposed model that accounts for most features of enzyme-catalyzed reactions. In this model the enzyme reversibly binds its substrate to form ES complex that subsequently breaks down to product
E + S ES E + P k1 k2
k-1
Where
E: enzyme
S: substrate
ES: enzyme-substrate complex
P: product
k1 k-1 k2 are rate constants
Michaelis-Menten Equation
The Michaelis-Menten Equation describes how reaction velocity varies with substrate concentration
V [S]
Rate v =
km + [S]

24. Assumptions Required for Michaelis-Menten Equation
E and S combine to form ES complex
There is only a single substrate, the concentration of S is much greater than concentration of enzyme the amount of the substrate bound by the enzyme at any one time is small.
E + S = ES rapidly reaches equilibrium: steady state assumption, concentration of ES is constant, dose not change with time the rate of formation of ES is equal to that of the breakdown of ES (to E and P)
Reaction from P is irreversible
only the initial reaction velocities are used in the analysis of enzyme reactions- the rate of reaction at zero time, at that time the concentration of eth product is very small  the rate of the back reaction from P to S can be ignored
Michaelis-Menten Equation
Vo: initial reaction velocity
Vmax: maimal velocity
Km: michaelis-menten constant= (k-1+K2)/K1
[S]: concentration of substrate
V max[S]
vo =
km + [S]

25. Michaelis-Menten Model

26. Michaelis-Menten Model

27. Conclusions about Michaelis-Menten kinetics
Characteristics of Km:
The Km constant is a characteristics of an enzyme and particular substrate
Km reflects the affinity of the enzyme for a particular substrate
Km numerically equal to the concentration substrate at which the reaction velocity is equal to ½ Vmax.
Km dose not vary with concentration of the enzyme
low Km  high affinity; low [S] is needed to half-saturate the enzyme
large Km  low affinity; high [S] is needed to half-saturate the enzyme

28. Relationship between the velocity to enzyme concentration: the rate of reaction is directly proportional to the enzyme concentration at all substrate conc. if the enzyme concentration is halved  the Vo is reduced to half
Order of reaction:
When [S] << Km, V (velocity of reaction) is roughly proportional to [S] the rate of the reaction is first order reaction
When [S] >> Km, the V is constant and equal Vmax. The rate of reaction is independent on the [S] the reaction rate is Zero order

29. Double reciprocal of Michaelis-Menten equation Lineweaver-Burk Plot
When V is plotted versus the [S], it is not always possible to determine the Vmax or Km from the graph because of gradual upward slope of the hyperbolic curve
vo =Vmax [S] / (Km + [S]) (Michaelis-Menten Equation)
Equation of straight line: y= ax + b (a = slope, b = y intercept
Linear Transform: take reciprocal of each side of equation
1/vo = Km/Vmax • 1/[S] + 1/V
y = a x b

30. Double reciprocal of Michaelis-Menten equation Lineweaver-Burk Plot

31. Lineweaver-Burk Double Reciprocal Plot
1/vo = Km/Vmax • 1/[S] + 1/Vmax
Lineweaver-Burk line allow us to determine the Vmax and Km simply
The x intercept is -1/km
The y intercept is 1/Vmax

32. What do we mean by inhibitors?

33. Enzyme Inhibition
Enzyme inhibitor: any substance that can reduce the velocity of an enzyme-catalyzed reaction and cause a loss of catalytic activity
Inhibitors can be reversible or irreversible.
- Reversible inhibitors bind to enzymes through a non-covalent bonds  Upon dilution the EI (enzyme-inhibitor) complex dissociate and recover the enzyme activity, may be competitive or noncompetitive
- Irreversible inhibition occur when the inhibited enzyme can not recover its activity by dilution. The inhibitors form a covalent bonds with the active site enzyme or destruction of the protein structure of the enzyme
Irreversible Inhibitors

34. Competitive inhibitors
This type of inhibition occurs when the inhibitors bind reversibly to the same site that the substrate normally occupy  compete the substrate for that site
A competitive inhibitor
Has a structure similar to substrate
Occupies active site
Competes with substrate for active site
Has effect reversed by increasing substrate concentration

35. Competitive inhibitors
Vmax: the effect of the competitive inhibitors is reversed by increasing the [S]. At sufficient high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of the inhibitors. The y-intersect is unchanged
Km: a competitive inhibitors increases the apparent Km for a given substrate in the presence of this inhibitors , more substrate is needed reach the Vmax. The x intersect is changed indicating that the apparent Km is increased

36. Non-Competitive Inhibitors
Inhibitor binds at a site distinct from the substrate site
It may bind to free E or to ES.
Once bound it will prevent P formation.
And the affinity of the I to both E and Es is the same.

37. Noncompetitive Inhibition
Non competitive inhibition occurs when the inhibitor at a site distinct from the substrate site
A noncompetitive inhibitor does not have a structure like substrate.
Alter the shape of enzyme and active site Substrate cannot fit altered active site
It binds either to the enzyme or to the ES complex No reaction occurs
Effect is not reversed by adding substrate
It may bind to free E or to ES. Once bound it will prevent P formation.
And the affinity of the I to both E and ES is the same.

38. Non-competitive inhibitors decrease the Vmax, and can not be overcome by increasing the substrate concentration
Non-competitive inhibitors don’t affect the Km, the enzyme show the same Km in the absence or the presence of the inhibitors

39. Enzyme inhibitors could be used as drugs
Different types of enzyme inhibitors
I 2
I 1
I 0
NONCOMP
COMP

40. Allosteric Enzymes and Allosteric Regulation
Allosteric enzymes: are regulated by molecules called effectors (modulators) that bind non-covalently at site other than the active site.
Allosteric enzymes show Sigmoidal curve and don't follow Michalaelis-menten rules
Allosteric modulators affect the enzyme affinity to its substrate and /or modify the maximal catalytic activity of the enzyme
Enzyme regulation is essential to coordinate the numerous metabolic processes
Most of enzymes respond to changes of substrate concentration  an increase in the substrate conc. lead to increase in the rate of enzyme action  return the substrate conc. to normal level
Some enzymes respond to allosteric effectors or covalent modification that affect the velocity of enzyme

41. Allosteric regulators (effectors)
-Effectors that inhibit enzyme activity are termed negative effectors while those increase the enzyme activity are called positive effectors
- Homotropic effectors:
The substrate itself acts as effector, usually allosteric substrate are positive effector.
The presence of substrate molecule at allosteric site will increase the catalytic activity of the substrate-binding site  sites cooperativity  Sigmoidal curve of V vs [S] not hyperbolic
Heterotropic effectors
The effectors are different from substrate
Feed back inhibition
A B C DE
This enzyme has allosteric site that can bind to E
The final product may have a feedback inhibition of on the enzyme that convert AB
Feed back inhibition serves to coordinate the flow of substrate molecules through a series of reactions with the need of the cell.

42. Enzymatic Regulation

43. Covalent modification-Protein phosphorylation
- Many enzymes may be regulated by covalent modification; addition or removal of phosphate group
Phsphorylation occurs at the –OH of serine, threonine or tyrosine residues
Phosphorylation process could be Activation or Deactivation process  depending on the enzyme itself
Inactive or
active

44. Induction and repression of enzyme synthesis
Cells can regulate the amount of eth enzyme present, usually by altering the rate enzyme synthesis
The induction or repression synthesis of protein leads to an alteration of the total number of enzyme population  increasing the number of active sites and Not affecting the enzyme activity.

45. The End

48. Learning Objectives
Gibbs Free Energy Diagram – illustrates activation energy and two chemical steps
Define Enzyme – protein, catalyst, specific, activates substrate
Binding of Substrate to Active Center
Transition State binds tighter than substrate or product
Mechanism of Chymotrypsin – DHS triad and tetrahedral intermediate, swinging His

49. Learning Objectives Lineweaver-Burk plot
Competitive, Uncompetitive and Noncompetitive Inhibition
Possible models of inhibition
Suicide and irreversible inhibition
Natural inhibitors

50. Competitive inhibitors

54. Uncompetitive Inhibitors
Inhibitor binds at an allosteric site, but only to the ES complex
The slopes of 1/Vo vs. 1/[S] are unchanged but Vmax is lower, and so is the apparent [S] needed to reach 1/2 Vmax = Km. Vo

55. Enzyme inhibitors could be used as drugs
Different types of enzyme inhibitors
COMP UNCOMP NONCOMP