1. Berg • Tymoczko • Stryer
Biochemistry
Chapter 8:
Enzymes:
Basic Concepts and Kinetics
Copyright © 2007 by W. H. Freeman and Company

2. The enzyme Aequorin catalalyzes the oxidation of this compd in presence of calcium to release CO2 and light

3. Properties of Enzymes
Enzymes are protein catalysts. Increase rate of chemical reaction
NOT CONSUMED.
Show high catalytic efficiency…enzyme catalyzed reactions from 103 to 106 times faster.
Highly specific, catalyze only one type of chemical reaction
Some have non-protein cofactor needed for enzymatic activity.
Cofactors commonly metal ions like Zn2+ and Fe2+, derivatives of vitamins, NAD+ , FAD, and Coenzyme A.

4. Terminology Holoenzyme = enzyme (protein) + cofactor (non protein)
absence of cofactor, no activity
Apoenzyme: protein portion of holoenzyme
Zymogen (Proenzyme): catalytically inactive enzyme complex (Ex: Chymotrypsinogen, pepsin)
Prosthetic group: tightly bound cofactor that does not dissociate from enzyme (Ex: Biotin).

5. Enzymes Powerful, Highly Specific Catalysts
Accelerate reactions by six orders of magnitude or more.
Carbonic anhydrase
catalyzes hydration of carbon dioxide
One of fastest enzymes known
Catalyzed reaction is 107 times as fast as uncatalyzed one

6. Enzymes Powerful, Highly Specific Catalysts
Precise interaction of substrate with enzyme
Three dimensional structure of enzyme
Specific in terms of
Reaction
Substrate
Catalyzes single chemical reaction or a set of closely related reactions
Eg. Proteases: catalyze hydrolysis of peptide bonds.
Most proteolytic enzymes also catalyze a different but related reaction in vitro
hydrolysis of an ester bond

7. Enzymes Are Powerful and Highly Specific Catalysts
Substrate specificity
Proteolytic enzymes differ markedly in their substrate specificity
Papain
Wide substrate specificity
Cleaves any peptide bond
Trypsin
specific
Cleaves only on the C terminus of Lysine and Arginine residues
Thrombin
Very Specific
Catalyzes hydrolysis of Arg-Gly bonds in a particular sequence only

8. Cofactors
Catalytic activity of enzymes depends on presence of small molecules, Cofactors
Apoenzyme + cofactor = holoenzyme
Cofactors
Metals
Small organic molecules: coenzymes
Derived from vitamins
Tightly bound termed prosthetic groups
Loosely bound like co-substrates

10. Free energy Useful Thermodynamic Function to Understand Enzymes
Key activity in living systems is ability to convert one form of energy into another
Photosynthesis
Light energy converted into chemical energy
Enzymes play vital roles in energy transformation
Play fundamental role in photosynthesis and cellular respiration
Other enzymes use chemical bond energy of ATP in diverse ways
Myosin converts bond energy of ATP into mechanical energy of contracting muscles

11. Free energy Useful Thermodynamic Function to Understand Enzymes
Thermodynamic properties of reaction
Free energy difference between reactants and products
Determines if reaction occurs or not
Energy to initiate conversion of reactants into products
Determines rate of reaction
Affected by enzymes

12. Bioenergetics Study of energy changes in biochemical reactions
Why some reactions occur while others do not.
Bioenergetics deals with energy:
Release
Storage
Use in biological systems
Nonbiological reactions utilize heat energy.
Biological systems isothermic, use chemical energy.

13. Free energy Useful Thermodynamic Function to Understand Enzymes
Free energy change (ΔG) of chemical process is measure of energy available to do work.
ΔG = Gproducts – Greactants
If ΔG <0 , forward reaction favored.
Products more stable than reactants. As reaction proceeds, energy released that can do work.
If ΔG = 0, reactants and products at equilibrium
If ΔG >0, reactants at lower energy than products; energy needed for reaction.
ΔG is independent of the path of the transformation
ΔG for oxidation of glucose the same whether combustion or enzyme catalyzed reactions
ΔG provides no information on rate of reaction. Rate depends on free energy of activation, unrelated to ΔG of reaction

14. Free energy Useful Thermodynamic Function to Understand Enzymes
Standard Free Energy Change is related to Equilibrium Constant
ΔGo’ is directly dependent on the equilibrium constant K’eq.
For a reaction:
A B C + D
ΔG = ΔGo’ + RT. ln [C] [D]/[A] [B]
ΔG depends on the nature of the reactants (expressed in the ΔGo’ term) as well as the concentration of the reactant and products
At equilibrium, ΔG = 0, and K’eq = [C] [D]/[A] [B]
ΔGo’ = - RT.ln K’eq or ΔGo’ = RT log K’eq
since R= x 10-3 kJ.mol-1.deg-1 and T = 298K,
ΔGo’ = log K’eq or K’eq = 10 –ΔGo’ /5.71
The free energy change of the forward reaction is equal in magnitude but opposite in sign to that of the backward reaction.
A  B -5 kcal
B  A +5 kcal

15. Important Facts
Criterion for reaction under specified conditions depends on DG not DG0. Knowing concentrations of reactants allows determination!
Standard free energy changes additive in any sequence of consecutive reaction:
As long as the sum of DGs of individual reactions is negative, the pathway can potentially proceed as written
Even if some of the individual component reactions of the pathway have +DGs.

16. Isomerization of DHAP to GAP
At 250C at equilibrium
[GAP]/[DHAP] =
ΔGo’ = - RT.ln K’eq
= X 10-3 X 298 X ln (0.0475)
= 7.53 kJ/mol
Reaction is endergonic and will not convert spontaneously
For initial concentration
For [DHAP]/[GAP] = 0.015
ΔG = ΔGo’ + RT.ln K’eq
= 7.53kJ/mol kJ/mol = -2.89kJ/mol
ΔG indicates it is exergonic and spontaneous
Criterion of spontaneity for a reaction is ΔG; not ΔGo’
Reactions not spontaneous based on ΔG can be spontaneous by
adjusting concentrations of reactants and products.

17. Additive property of free energy change
Two or more reactions coupled when products of one reaction are reactants of next reaction.
Multiple reactions coupled, become a pathway.
Pathway must satisfy minimally two criteria:
The individual reactions must be specific, yielding only one particular product or set of products.
The entire set of reactions in a pathway must be thermodynamically favored
An important thermodynamic fact: the overall free energy change for a chemically coupled series of reactions is equal to the sum of the free-energy changes of the individual steps
As long as the sum of ΔG’s of the individual reactions is negative the pathway is thermodynamically favorable.
A  B + C G0’ = + 5 kcal mol-1
B  D G0’ = - 8 kcal mol-1
*******************************
A  C + D G0’ = - 3 kcal mol-1

18. Enzymes Only alter reaction rate Cannot alter reaction equilibrium
Eg. Conversion of S to P
kF = 10-4 s-1 and kR = 10-6 s-1
K = [P]/[S]=kF/kR = 100
Equilibrium concentration of P is 100 times that of substrate, irrespective of enzyme
Might take a very long time to achieve this equilibrium in absence of enzyme
Equilibrium attainted rapidly in presence of suitable enzyme
Thus, enzymes accelerate attainment of equilibrium but do not shift positions.

19. How Enzymes Work
Accelerate reactions by facilitating formation of the transition state
Chemical reaction of substrate (S) to form product (P) goes through transition state (X‡)
S X‡ P
Transition state (X‡):
Top of the energy hill;
Highest free energy, least stable
May go to S or P.
Not reaction intermediate
Bond breakage, bond formation, charge developments take place.
Difference in free energy between transition state and substrate called “Gibbs free energy of activation” or “activation energy”
G‡ = Gx‡ - Gs
V (reaction rate) proportional to  G‡
 G‡ does not enter  G calculation as energy required to generate this is released during product formation.
This is how enzymes alter reaction rate without altering  G of reaction
Enzymes function to lower activation energy or facilitates formation of transition state

20. How Enzymes Work Enzymes form ES complex.
Existence of ES complexes shown in many ways:
At constant concentration of enzyme, reaction rate increases with increase in [S] until a Vmax is reached.
Maximal velocity suggests formation of ES complex
X-ray crystallography
Images of substrate analogs bound to active sites
Spectroscopic characteristics of many enzymes and substrate change upon formation of ES complex.

21. Active Site
Region that binds substrate and possesses catalytic residues that participate in reaction mechanism.
Interaction of enzyme and substrate at active site promotes formation of transition state.
Region that lowers activation energy of a reaction, thus speeds up reaction

22. Active Site
Although structure, specificity, or mode of catalysis of enzymes differ, their active sites share common features
Three dimensional cleft, or crevice, formed by groups from different parts of amino acid sequence
Small part of total volume of enzyme
Enzymes large three-dimensional structures
Serve as scaffold to create 3D active site
Constitute regulatory sites
Interaction with other proteins
Substrate channels
Unique Microenvironments
Water usually excluded unless a reactant
Non polar environment enhances binding of substrates as well as catalysis.
Polar residues acquire special properties essential for substrate binding and catalysis in microenvironment.

23. Structure of enzyme-substrate complex

24. Active sites include distant residues

25. Active Site
Substrates bound to enzymes by multiple weak reversible interactions
Electrostatic
Hydrogen bonds
Directional character results in high degree of specificity
Van der Waals forces
Becomes significant when numerous substrate atoms come close to many enzyme atoms which requires enzyme and substrate to have complementary shapes.

26. The specificity of binding depends on the precisely defined arrangement of atoms in an active site.
Since enzymes and substrates interact by means of short range forces that require close contact, a substrate must have a matching shape to fit into the site.
Emil Fisher’s Lock and Key model (1890)
Enzymes are flexible and shape of the active site can be markedly modified by the binding of substrate
Daniel Koshland Jr.’s dynamic induced fit model (1958)
Active site assumes a shape that is complementary to that of the substrate only after the substrate has been bound.

27. Induced-Fit Model
In absence of substrate, catalytic and substrate binding groups are far from each other.
In presence of substrate, conformational change occurs in the enzyme… results in
Aligning groups correctly for substrate binding and catalysis
Substrate analogs cause some, not all correct conformational changes.
Active site has shape complementary to that of substrate only after substrate is bound.

28. Enzyme-Substrate binding energy
Binding energy: Free energy released upon multiple weak interactions between enzyme and substrate.
Maximized when right substrate interacts to form interactions
Accounts for substrate specificity
Maximal energy released when enzyme facilitates formation of transition state
Binding energy lowers activation energy
Transition state forms S or P depending reaction ΔG

29. Definition of Terms
Substrate: substance acted upon by an enzyme. Activity: amount of substrate converted to product by enzyme per unit time (M/min). Specific activity: activity per quantity of protein (M/min/mg protein). International unit: quantity of enzyme needed to transform 1.0 micromole of substrate to product per minute at 30C and optimal pH.

30. The Michaelis-Menten Equation Describes the Kinetic Properties of Many Enzymes

35. Meaning of KM Two meanings: Measure of strength of ES complex
Substrate concentration when half the active sites filled
Dissociation constant of ES complex when k2 is small
Measure of strength of ES complex
High KM  weak binding
Low KM  strong binding

38. Importance of KM CH3CH2OH + NAD+  CH3CHO + H+ + NADH
Some individuals sensitive to ethanol, exhibit facial flushing, rapid heart rate, after ingesting alcohol. In the liver:
Alcohol
dehydrogenase
CH3CH2OH + NAD+  CH3CHO + H+ + NADH
Aldehyde
CH3CHO + NAD+ + H2O  CH3COO- + NADH + 2H+

39. Most people have 2 forms of acetaldehyde dehydrogenase:
low Km, mitochondrial form
high Km, cytosolic form
In susceptible persons:
the mitochondrial enzyme less active because single amino acid substituted and
acetaldehyde processed only by cytosolic enzyme.
Cytosolic enzyme has high Km,
less acetaldehyde converted into acetate,
increased acetaldehyde goes to blood, and
symptoms appear.

40. kcat
Turnover number:
Number of S molecules converted into product by enzyme molecule in a unit time when the enzyme fully saturated with substrate.
It is equal to the k3.
k3= Vmax/ET

42. kcat/KM kcat/ KM can be used as a measure of catalytic efficiency.
When the [S]>>>>KM, the rate of catalysis is equal to kcat.
But, under physiological conditions, the [S]/KM is equal to (meaning [S]<<<<KM)
The enzymatic rate is less than kcat because enzymes active sites are unoccupied.
kcat/ KM can be used as a measure of catalytic efficiency.
By using kcat/ KM, we can compare an enzyme’s preference for different substrates.

44. Most biochemical reactions include multiple substrates.
Multiple substrate reactions can be divided into 2 classes:
Sequential displacement
Double displacement
Lactate dehydrogenase enzyme: ordered sequential (NADH must bind first and lactate released first)

45. The enzyme (LDH) exists as a ternary complex
Sequential displacement (ordered)
The enzyme (LDH) exists as a ternary complex

46. Sequential displacement (random)
The order of addition of substrates and release of products is random! Creatine kinase is the enzyme.

47. Cleland notation

48. Double -displacement (ping-pong) reactions
One or more products are released before all substrates bind the enzyme.
Enzyme: aspartate amino transferase

49. Ping-pong mechanism

50. Allosteric enzymes Do not obey M-M model!
Multiple subunits, multiple active sites.
Display sigmoidal plots.
Binding of S to one active site affects properties of other active sites in same enzyme molecule.

51. Allosteric Models:

52. Allosteric inhibitor stabilizes the “T” state whereas an allosteric activator stabilizes the “R” state.

54. Allosteric activators and inhibitors shift the curve in different directions (fES: fraction of sites filled)

55. Enzymes inhibited by specific molecules
Important because:
Major control mechanism
Many drugs and toxic agents act this way.
2 kinds of enzyme activity:
Reversible
competitive
noncompetitive
Irreversible
I dissociates very slowly from the enzyme because I binds to the enzyme covalently. (Ex.: nerve gases)

60. Enzyme inhibitors Reversible Irreversible Competitive Noncompetitive
Group specific reagents
Substrate analogs
Suicide inhibitors

67. Competitive inhibitors
Resemble substrate, bind to active site of enzyme
Methotrexate is structural analog of tetrahydrofolate(coenzyme of dihydrofolate reductase that is involved in purine and prymidine synthesis)
Diminish rate of catalysis by reducing proportion of enzyme molecules bound to substrate
Competitive inhibitors are used suchmmmmmmmm

69. Irreversible inhibitors used to map active site
Functional groups important for enzymes activity.
How can we be certain about functional groups?
X-ray crystallographic of enzyme bound to its substrate
Irreversible inhibitors provide alternative approach

70. Three groups of irreversible inhibitors
Group specific
Reacts with R groups of amino acids
DIPF(diisopropylphosphofluoridate)
Iodoacetamide
Substrate analog
Molecules structurally similar to S for enzyme that covalently modifies active site
TPCK (tosyl-L-Phe chloromethyl ketone)
3 bromoacetol
Suicide inhibitors
Enzyme participates in its own irreversible inhibition

72. Iodoacetamide can inactivate an enzyme by reacting
with a critical cysteine residue.

76. Mechanism-based (suicide) inhibition (MAO inhibitor)

80. Suicide inhibitor of MAO

81. Transition state analogs are potent inhibitors of enzymes

84. Penicillin irreversibly inactivates key enzyme in bacterial cell wall synthesis
Cell wall made of macromolecule called “peptidoglycan”, linear polysaccaride chains crosslinked by short peptides.
Penicillin blocks last step of cell wall synthesis, i.e., crosslinking of different peptidoglycan strands.
Penicillin inhibits crosslinking action of “glycopeptide transpeptidase” enzyme.
Penicillin fits in active site of enzyme because looks like D-Ala-D-Ala!

85. Structure of penicilline

87. Peptidoglycan in staphylococcus:
sugar yellow
peptide red
pentagly blue

88. Formation of crosslinks in peptidoglycan
The terminal amino group of the pentaglycine bridge attacks the peptide bond.

89. Transpeptidation reaction
An acyl-enzyme intermediate is formed in the transpeptidation reaction leading to cross-link formation.

92. Conformations of penicillin and a normal substrate
Penicillin resembles the postulated conformation of the transition state of D-Ala-D-Ala

93. Formation of a penicilloyl-enzyme complex