ME02 - ENZYMES : Introduction Flashcards
Energy required in order for reaction to occur
Activation Energy
Determine the direction and equilibrium states of the reaction
Free energy changes
Catalysts
Enzymes
Properties of Enzymes
Reaction-specific
Substrate-specific
Stereo-specific
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
Non-protein catalysts with ribonuclease & peptidyl transferase activity
Ribozymes
What kind of gene is present in Ribozymes and its function
It contains autocatalytic RNA molecules that can adopt complex structures like proteins
Involved in Gene Therapy
Intron and tRNA processing
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
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
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
Example of Oxidoreductase
Lactic acid-dehydrogenase - oxidizes lactic acid to form pyruvic acid during FERMENTATION
Moving a functional group from one substrate/molecule to another
(may be anabolic)
Transferase
Example of Transferase
Hexokinase - transfers phosphate from ATP to glucose in the first step of glycolysis
Break single bonds (ester, ether, peptide or glycosidic) by the addition of water
This is Catabolic
Hydrolysis
Example of Hydrolysis
Lipase - breaks down lipid molecules
Form or cleave bonds with group elimination non hydrolytically
Splitting a chemical into smaller parts without using water
Lyase
How does LYASE catalyze cleavage of C-C, C-O, C-N, and other covalent bonds
By atom elimination and generating double bonds
Example of Lyase
Fructose 1,6-bisphosphate aldolase - splits fructos into G3P and DHAP
Carry out intramolecular rearrangements
Catalyze geometric or structural changes within a molecule
(Neither catabolic or anabolic)
Isomerase
Example of Isomerase
Phosphoglucoisomerase - converts glucose 6 phosphate into fructose 6 phosphate during glycolysis
Link two substrates together usually with the Hydrolysis of ATP
Joining two or more chemicals together coupled with ATP hydrolysis
Ligase or Polymerase
Example of Ligase/Phosphorylase
Acetyl-CoA synthetase - combines acetate and coenzyme A to form acetyl-CoA for the Krebs Cycle
ENZYMES THAT HAS Catabolic Reactions
Hydrolases - Lipase
Lyase - Fructose 1,6-Bisphosphate aldolase
ENZYMES THAT HAS Anabolic Reactions
Transferase - Hexokinase
Ligase/Polymerase - AcetylCoA synthetase
ENZYMES THAT HAS Neither Catabolic and Anabolic Reactions
Isomerase - Phosphoglucoisomerase
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
Events happening on the Active Site of Enzyme
Desolvation effects
Binds substrates properly for transition state formation
Binds cofactors & prosthetic groups
Factors for substrates’ transition state formation properly
Geometric & Electronic complementarity
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
Reciprocal changes in both substrate & enzyme structure binding
Hand glove fitting
Induced-fit model by Daniel Koshland
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
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
Components of active holoenzyme
Inactive apoenzyme & the non-protein cofactor
Participate in substrate binding or in catalysis
Molecules that are required by certain enzymes to carry out analysis
Co-factors
Binds to the active site of enzyme and participate in catalysis but are not considered substrates of the reaction
Co-factors
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
Co-enzymes that participate in oxidation-reduction reaction
Nicotinamide
Flavin
Non-Vitamins are Tetrahydrobiopterin & Ubiquinone
Needed in Kinase-Catalyzed reactions
Presents “Charge shielding”
Nucleoside triphosphates
Mg2+
Involved in electron-transfer reactions
Iron in heme
Iron-sulfur bridges
How does enzymes form transition states at a lower activation energy
Through strategic binding & Catalytic Residues/Cofactors
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
Promotes catalysis through charge stabilization and water ionization, acting as Lewis “super” acids
Metal Ion Catalysis
How does proteases work in forming an unstable tetrahedral intermediate
Covalent
Acid-base Catalysis
What is the similarity in covalent and non covalent (acid base catalysis) processes for proteases
Stabilization of the tetrahedral intermediate
Nucleophiles that participate in covalent catalysis
Serine, Cysteine or Threonine
Base is usually Histidine
Acids and Bases involved in General Acid-Base Catalysis
Side chains of aspartic residues or glutamic residues
Zinc in case of metalloproteinases
Fundamental Distinction between the covalent catalysis and general acid-base catalysis
Evolution of natural inhibitors, chemistries available for design of small molecule inactivators
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
How does serine proteases display bond specificity
Through their active site pockets
How are serine proteases synthesized and activated
They are synthesized as zymogens.
They are activated via proteolysis by another serine protease or by autolysis
Pockets of Serine Proteases
Trypsin - deep narrow pocket with Asp
Chymotrypsin - wide hydrophobic pocket
Elastase - very shallow, narrow pocket
Serine proteases that are degradative proteases of Digestive System
Trypsin, Chymotrypsin, Elastase
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
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
Serine proteases that are degradative bacterial protease, sometimes added to laundry detergents to break down protein-pigment complexed in blood and grass stains
Substillin
What part does deprotonated serine side chain attack to produce tetrahedral oxyanion intermediate
Carbonyl Carbon
Involves stabilization of tetrahedral intermediate states through hydrogen bonds
What is donated by the protonated histidine, acting as a general acid, to generate quaternary amine
Donating a proton to the amino group
What results in Proteolysis
Quaternary amine & Tetrahedral oxyanion collapse
What deprotonates Histidine then attacks the carbonyl carbon for the formation of another oxyanion intermediate
Histidine deprotonates H2O
When tetrahedral oxyanion collapses, what happens.
It liberates the peptide & regenerating serine
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
Rate of enzyme-catalyzed reactions in humans generally doubles with every ______________
increase of 10˚C until 45-55˚C
All enzyme-catalyzed reactions depend on ___________
Optimal Hydrogen Ion reaction
Factors affecting reaction rates
Temperature
pH (Hydrogen Ion Concentration)
Substrate concentration
Related to the ionization of specific amino acid residues that constitute the substrate binding site
Optimum pH
What kind of graph is produced in substrate concentration vs reaction rate
Rectangular Hyperbolic curve
Rectangular Hyperbolic Curve represents _________
Plateauing towards enzyme saturation
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
The substrate concentration at half the maximal velocity
Michaelis contant Km
In Michaelis Menten Plot, when is Vmax approached
Vmax is approached when [S] is close to 20 km
What does the Michaelis constant approximate
Binding constant
When [S] is less than, equal to or greater than Km, what is the effect on V?
At [S] > Km, V ≈ Vmax
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
Allows precise determination of Km & Vmax at less than saturating concentrations
Double reciprocal or Lineweaver-Burk plot
Alternative single-reciprocal plots
Eadie-Hofstee
Hanes-Woolf plots
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
Compares the relative activity of enzymes
Specificity Activity
Turnover Number
Catalytic Constant
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
To compare across homogenous enzymes
Turnover Number
Vmax/mol(enzyme)
Larger the turnover number = faster reaction
S(t) = number of active sites
Unit = s-1
Best expressed in the ratio kcat/km
Catalytic Constant (Km)
Describes the behavior of enzymes exhibiting cooperativity
Hill equation
Depicts cooperativity in multimeric enzymes
Hill Plots
Sequential reactions
Any of the substrates may combine first followed by the other substrate before catalysis can begin
Random sequential reactions
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
One or more products are released before all substrates are added
Double displacement reactions
Kind of Lineweaver Burk plots displacement reactions produce
Single Displacement Reaction - Intersecting Lineweaver Burk plot
Double Displacement Reaction - Parallel Lineweaver Burk plot
All substrates must combine with the enzymes before a reaction can occur & products can be released.
Single Displacement Reactions
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
Alters the structure of an enzyme and thus also change its function
Inhibitors
Type of Inhibitor
Denaturation
Examples are Acids & Bases, Temperature, Alcohol, Heavy Metals, Reducing Agents
Non-Specific
Type of Inhibitor
Irreversible
Reversible - Competitive
- Non competitive, allosteric, feedback
Specific
Potent inhibitors
Compounds with a structure that resemble the transition state of a substrate
Transition State Analogs
Enzymes that interact with a substrate by means of ______________, moving the substrate towards the transition stte
By means of strains or distortions
Enzymes inhibitors which resemble the transition state structure would ______
Bind more tightly to the enzyme than the actual substrate
How can transition state analogs be able to be used as inhibitors
By blocking the active site of the enzyme
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
Substrate analogs that bind to the active site, preventing enzyme-substrate complex formation
Competitive inhibitors
Bind to the free enzyme & enzyme substrate complex at the allosteric site and lower the cat efficiency of the enzyme
Simple noncompetitive
Bind to the enzyme-substrate complex rather tan to the free enzyme and lower both Vmax and Km
Uncompetitive inhibitors
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
Facilitates the evaluation of inhibitors
Double reciprocal plots
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
Endergonic vs Exergonic Reaction
Endergonic Exergonic
non-spontaneous spontaneous
energy is required energy is required
activation energy is higher
