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1

Energy required in order for reaction to occur

Activation Energy

2

Determine the direction and equilibrium states of the reaction

Free energy changes

3

Catalysts

Enzymes

4

Properties of Enzymes

Reaction-specific
Substrate-specific
Stereo-specific

5

Description for Enzymes

Increase reaction rates without being consumed or permanently altered
D sugars & L-amino acids
Typically proteins but can also be nucleotides
Affected by pH and temp

6

Non-protein catalysts with ribonuclease & peptidyl transferase activity

Ribozymes

7

What kind of gene is present in Ribozymes and its function

It contains autocatalytic RNA molecules that can adopt complex structures like proteins

8

Involved in Gene Therapy

Intron and tRNA processing

9

Enzymes classified by reaction. Complete table
Class Type of Reaction Example
Hydrolase
Isomerase
Ligase/Polymerase
Lyase
Oxidoreductase
Transferase

Class Type of Reaction Example
Hydrolase Hydrolysis Lipase

Isomerase Rearrangement of atom Phosphoglucoisomerase
within a molecule

Ligase/Polymerase Joining two or more Acetyl-CoA synthetase
chemicals together

Lyase Splitting a chemical into Fructose 1,6-BP Aldolase
smaller parts w/o using water

Oxidoreductase Transfer of electrons or Lactic acid
H atoms from one molecule dehydrogenase
group to another

Transferase Moving a functional group Hexokinase
from one molecule group to another

10

IUBMB Number corresponds to
1st number
2nd number
3rd number
4th number

1st number - Major class: Enzymes
2nd number - Subclass: Mechanism
3rd number - Sub-Subclass: Substrate Clase
4th number - Specific Substrate

11

Catalyze the oxidation of a substrate with simultaneous reduction of another substrate or coenzyme
Transfer of electrons or H atoms from one molecule to another

Oxidoreductases

12

Example of Oxidoreductase

Lactic acid-dehydrogenase - oxidizes lactic acid to form pyruvic acid during FERMENTATION

13

Moving a functional group from one substrate/molecule to another
(may be anabolic)

Transferase

14

Example of Transferase

Hexokinase - transfers phosphate from ATP to glucose in the first step of glycolysis

15

Break single bonds (ester, ether, peptide or glycosidic) by the addition of water
This is Catabolic

Hydrolysis

16

Example of Hydrolysis

Lipase - breaks down lipid molecules

17

Form or cleave bonds with group elimination non hydrolytically
Splitting a chemical into smaller parts without using water

Lyase

18

How does LYASE catalyze cleavage of C-C, C-O, C-N, and other covalent bonds

By atom elimination and generating double bonds

19

Example of Lyase

Fructose 1,6-bisphosphate aldolase - splits fructos into G3P and DHAP

20

Carry out intramolecular rearrangements
Catalyze geometric or structural changes within a molecule
(Neither catabolic or anabolic)

Isomerase

21

Example of Isomerase

Phosphoglucoisomerase - converts glucose 6 phosphate into fructose 6 phosphate during glycolysis

22

Link two substrates together usually with the Hydrolysis of ATP
Joining two or more chemicals together coupled with ATP hydrolysis

Ligase or Polymerase

23

Example of Ligase/Phosphorylase

Acetyl-CoA synthetase - combines acetate and coenzyme A to form acetyl-CoA for the Krebs Cycle

24

ENZYMES THAT HAS Catabolic Reactions

Hydrolases - Lipase
Lyase - Fructose 1,6-Bisphosphate aldolase

25

ENZYMES THAT HAS Anabolic Reactions

Transferase - Hexokinase
Ligase/Polymerase - AcetylCoA synthetase

26

ENZYMES THAT HAS Neither Catabolic and Anabolic Reactions

Isomerase - Phosphoglucoisomerase

27

Where does Catalysis occur
The site on the enzyme where the substrate binds to

at the ACTIVE site
Active Site - cleft or pocket on the enzyme

28

Events happening on the Active Site of Enzyme

Desolvation effects
Binds substrates properly for transition state formation
Binds cofactors & prosthetic groups

29

Factors for substrates' transition state formation properly

Geometric & Electronic complementarity

30

Substrate-binding sites are largely preformed but some degree of induced-fit usually occurs on

Lock & Key model by Emil Fischer
Induced Fit model by Daniel Koshland

31

Reciprocal changes in both substrate & enzyme structure binding
Hand glove fitting

Induced-fit model by Daniel Koshland

32

What happens to the concentration rate in Induced Fit Model

Interactions that preferentially bind the transition state increase its concentration and therefore proportionally increase the reaction rate

33

What happens in the "glove-fitting" in Induced-Fit Model

The enzyme in turn induces a reciprocal changes in substrates, harnessing the energy of binding to facilitate the transformation of substrates into products

34

Components of active holoenzyme

Inactive apoenzyme & the non-protein cofactor

35

Participate in substrate binding or in catalysis
Molecules that are required by certain enzymes to carry out analysis

Co-factors

36

Binds to the active site of enzyme and participate in catalysis but are not considered substrates of the reaction

Co-factors

37

Vitamin B as precursors its coenzymes and Reaction Type
Vitamin B CoEnzyme Reaction Type
B1 Thiamine
B2 Riboflavin
B3 Panthotenate
B6 Pyridoxine
B12 Cobalamin
Niacin
Folic Acid
Biotin

Vitamin B CoEnzyme Reaction Type
B1 Thiamine TPP Oxidative phosphorylation
of alphaketo acids
B2 Riboflavin FMN, FAD Oxidoreduction
B3 Panthotenate CoA Acyl group transfer
B6 Pyridoxine PLP AA transamination & decarboxylation
B12 Cobalamin Methylcobalamin Isomerization (1C transfer)
Niacin NADP Oxidoreduction
Folic Acid Tetrahydrofolate 1 C group transfer
Biotin Biocytin Carboxylation

38

Co-enzymes that participate in oxidation-reduction reaction

Nicotinamide
Flavin
Non-Vitamins are Tetrahydrobiopterin & Ubiquinone

39

Needed in Kinase-Catalyzed reactions
Presents "Charge shielding"

Nucleoside triphosphates
Mg2+

40

Involved in electron-transfer reactions

Iron in heme
Iron-sulfur bridges

41

How does enzymes form transition states at a lower activation energy

Through strategic binding & Catalytic Residues/Cofactors

42

Factors in which enzymes bind substrates in a manner that favors bond formation

Proximity & Orientation
-High substrate concentration
-Proper alignment, low entropy

Catalysis by Strain
-Bonds become distorted, weak & prone to cleavage

43

Promotes catalysis through charge stabilization and water ionization, acting as Lewis "super" acids

Metal Ion Catalysis

44

How does proteases work in forming an unstable tetrahedral intermediate

Covalent
Acid-base Catalysis

45

What is the similarity in covalent and non covalent (acid base catalysis) processes for proteases

Stabilization of the tetrahedral intermediate

46

Nucleophiles that participate in covalent catalysis

Serine, Cysteine or Threonine
Base is usually Histidine

47

Acids and Bases involved in General Acid-Base Catalysis

Side chains of aspartic residues or glutamic residues
Zinc in case of metalloproteinases

48

Fundamental Distinction between the covalent catalysis and general acid-base catalysis

Evolution of natural inhibitors, chemistries available for design of small molecule inactivators

49

What is the Catalytic Triad

Catalytic Triad: Charge Relay Network
Serine - strong nucleophile that could attack carbonyl C
Histidine - accepts proton from Serine
Aspartate - stabilizes protonated Histidine

50

How does serine proteases display bond specificity

Through their active site pockets

51

How are serine proteases synthesized and activated

They are synthesized as zymogens.
They are activated via proteolysis by another serine protease or by autolysis

52

Pockets of Serine Proteases

Trypsin - deep narrow pocket with Asp
Chymotrypsin - wide hydrophobic pocket
Elastase - very shallow, narrow pocket

53

Serine proteases that are degradative proteases of Digestive System

Trypsin, Chymotrypsin, Elastase

54

Serine proteases that are regulatory proteases found in amplification cascades associated with blood clotting (thrombogenesis) or the dissolving of blood clots (thrombolysis) - opposing processes that regulate hemostasis

Plasmin, Tissue plasminogen activator, Thrombin

55

Serine proteases that are regulatory proteases that funcitn to activate peptide pro-hormones and growth factors by cleaving prosequences from the zymogen forms of such peptides

Kallikreins

56

Serine proteases that are degradative bacterial protease, sometimes added to laundry detergents to break down protein-pigment complexed in blood and grass stains

Substillin

57

What part does deprotonated serine side chain attack to produce tetrahedral oxyanion intermediate

Carbonyl Carbon
Involves stabilization of tetrahedral intermediate states through hydrogen bonds

58

What is donated by the protonated histidine, acting as a general acid, to generate quaternary amine

Donating a proton to the amino group

59

What results in Proteolysis

Quaternary amine & Tetrahedral oxyanion collapse

60

What deprotonates Histidine then attacks the carbonyl carbon for the formation of another oxyanion intermediate

Histidine deprotonates H2O

61

When tetrahedral oxyanion collapses, what happens.

It liberates the peptide & regenerating serine

62

SUMMARY SUMMARY SUMMARY

ENZYMES - Highly efficient & specific catalysts
Ribozymes - catalytic RNA molecules
Enzymes are classified based on 6 reaction types
Active site - binds/shields substrates & cofactors
Cofactors include co-enzymes, derived mostly from Vit B, metal ions and co-substrates
Enzymes catalyze reactions by proximity & strain and metal ion catalysis, acid-base catalysis and covalent catalysis

63

Rate of enzyme-catalyzed reactions in humans generally doubles with every ______________

increase of 10˚C until 45-55˚C

64

All enzyme-catalyzed reactions depend on ___________

Optimal Hydrogen Ion reaction

65

Factors affecting reaction rates

Temperature
pH (Hydrogen Ion Concentration)
Substrate concentration

66

Related to the ionization of specific amino acid residues that constitute the substrate binding site

Optimum pH

67

What kind of graph is produced in substrate concentration vs reaction rate

Rectangular Hyperbolic curve

68

Rectangular Hyperbolic Curve represents _________

Plateauing towards enzyme saturation

69

What happens to the rate of the enzyme at zero-order kinetics

The rate depends on how fast the product dissociates from the enzyme so that the latter may combine with more substrate

70

The substrate concentration at half the maximal velocity

Michaelis contant Km

71

In Michaelis Menten Plot, when is Vmax approached

Vmax is approached when [S] is close to 20 km

72

What does the Michaelis constant approximate

Binding constant

73

When [S] is less than, equal to or greater than Km, what is the effect on V?

At [S] > Km, V ≈ Vmax

74

What is the effect of decreasing the enzyme concentration

A large Km may result either from
K2 Product is formed rapidly
K1 Enzyme-substrate complex dissociates rapidly, suggesting low substrate affinity

75

Allows precise determination of Km & Vmax at less than saturating concentrations

Double reciprocal or Lineweaver-Burk plot

76

Alternative single-reciprocal plots

Eadie-Hofstee
Hanes-Woolf plots

77

Parameters Eadie-Hofstee Hanes Woolf
x-axis
y-axis
slope
x-intercept
y-intercept

Parameters Eadie-Hofstee Hanes Woolf
x-axis V [S]
y-axis V/[S] [S]/V
slope -1/Km 1/Vmax
x-intercept Vmax -Km
y-intercept Km/Vmax

78

Compares the relative activity of enzymes

Specificity Activity
Turnover Number
Catalytic Constant

79

Compares impure preparations of the same enzyme
Measures enzyme homogeneity and purity in body tissues and fluids: Maximal when all protein present is enzyme protein

Specificity Activity
Vmax/Protein

80

To compare across homogenous enzymes

Turnover Number
Vmax/mol(enzyme)
Larger the turnover number = faster reaction

81

S(t) = number of active sites
Unit = s-1
Best expressed in the ratio kcat/km

Catalytic Constant (Km)

82

Describes the behavior of enzymes exhibiting cooperativity

Hill equation

83

Depicts cooperativity in multimeric enzymes

Hill Plots

84

Sequential reactions
Any of the substrates may combine first followed by the other substrate before catalysis can begin

Random sequential reactions

85

Sequential reactions
One substrate must bind first with the enzyme to form a complex before the other substrate can bind and catalysis can begin

Ordered sequential reactions

86


One or more products are released before all substrates are added

Double displacement reactions

87

Kind of Lineweaver Burk plots displacement reactions produce

Single Displacement Reaction - Intersecting Lineweaver Burk plot
Double Displacement Reaction - Parallel Lineweaver Burk plot

88

All substrates must combine with the enzymes before a reaction can occur & products can be released.

Single Displacement Reactions

89

SUMMARY ON ENZYME KINETICS
Temperature
Michaelis-Menten equation
Lineweaver - Burk
Kcat/Km
Hill plots
Cooperative Binding

Temperature, pH & [Substrate] AFFECT reaction rates.
Michaelis-Menten equation GIVES the reaction rate
Lineweaver - Burk plots clearly SHOW THE VMAX AND KM
Kcat/Km - is the best measure of CATALYTIC EFFICIENCY
Hill plots - depict cooperativity in multimeric enzyme
Cooperative Binding -appears to be sequential (KNF)
Most enzymatic reactions are of the Bi-Bi type

90

Alters the structure of an enzyme and thus also change its function

Inhibitors

91

Type of Inhibitor
Denaturation
Examples are Acids & Bases, Temperature, Alcohol, Heavy Metals, Reducing Agents

Non-Specific

92

Type of Inhibitor
Irreversible
Reversible - Competitive
- Non competitive, allosteric, feedback

Specific

93

Potent inhibitors
Compounds with a structure that resemble the transition state of a substrate

Transition State Analogs

94

Enzymes that interact with a substrate by means of ______________, moving the substrate towards the transition stte

By means of strains or distortions

95

Enzymes inhibitors which resemble the transition state structure would ______

Bind more tightly to the enzyme than the actual substrate

96

How can transition state analogs be able to be used as inhibitors

By blocking the active site of the enzyme

97

Substrate analogs transformed by the catalytic machinery of the enzyme into a product that blocks the function of the same catalytic subunit

Suicide or mechanism based inhibitors

98

Substrate analogs that bind to the active site, preventing enzyme-substrate complex formation

Competitive inhibitors

99

Bind to the free enzyme & enzyme substrate complex at the allosteric site and lower the cat efficiency of the enzyme

Simple noncompetitive

100

Bind to the enzyme-substrate complex rather tan to the free enzyme and lower both Vmax and Km

Uncompetitive inhibitors

101

Lower concentration of S is required to form half of the ____________

Maximal concentration of ES, resulting in a reduction of the apparent value of KM

102

Facilitates the evaluation of inhibitors

Double reciprocal plots

103

Summary
Inhibitors
Transition state analogs
Enzyme
Competitive inhibitors
Uncompetitive inhibitors

Inhibitors alter the enzyme structure & thus funciton
Transition state analogs are potent inhibitors
Enzymes commit suicide through mechanism based inhibitors
Competitive inhibitors block active sites & increase Km
Noncompetitive inhibitors bind at allosteric sites & lower the Vmax
Uncompetitive inhibitors bind ES complexes and lower both Km and Vmax

104

Endergonic vs Exergonic Reaction

Endergonic Exergonic
non-spontaneous spontaneous
energy is required energy is required
activation energy is higher