ME02 - ENZYMES : Introduction Flashcards by Kate Alyssa Caton

Energy required in order for reaction to occur

Activation Energy

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Determine the direction and equilibrium states of the reaction

Free energy changes

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Catalysts

Enzymes

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Properties of Enzymes

Reaction-specific
Substrate-specific
Stereo-specific

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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

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Non-protein catalysts with ribonuclease & peptidyl transferase activity

Ribozymes

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What kind of gene is present in Ribozymes and its function

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

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Involved in Gene Therapy

Intron and tRNA processing

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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

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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

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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

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Example of Oxidoreductase

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

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

Transferase

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Example of Transferase

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

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

Hydrolysis

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Example of Hydrolysis

Lipase - breaks down lipid molecules

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

Lyase

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

By atom elimination and generating double bonds

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Example of Lyase

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

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

Isomerase

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Example of Isomerase

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

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

Ligase or Polymerase

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Example of Ligase/Phosphorylase

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

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ENZYMES THAT HAS Catabolic Reactions

Hydrolases - Lipase

Lyase - Fructose 1,6-Bisphosphate aldolase

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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

ME02 - ENZYMES : Introduction Flashcards

(104 cards)