Enzymes and Enzyme Kinetics Flashcards
What are the characteristics of enzymes?
1) Enzymes increase the rate(s) of a reaction; enzymes can accelerate rxn’s as much as 1016 over uncatalyzed rxn’s
2) Enzymes are not consumed / destroyed during a chemical rxn
3) Enzymes may be localized to specific areas
4) Enzymes do NOT change the energetics of a reaction. That is, they do not change the delta G of a reaction.
5) Enzymes are present at low concentrations
6) Enzymes increase the probablity of a rxn occuring and therefore increase the rate of a rxn
7) Enzyme activity can be regulated
Describe the physical appearance of an enzyme.
Enzymes are large, multi-subunit proteins. Their structure is vital to maintaining the integrity of he active site of the enzyme.
Describe the active site of the enzyme.
The structure of an enzyme may serve to channel the substrate to the active sites binding residues.
The active site may contain cofactors or coenzymes that are essential for catalytic action.
What is a cofactor?
A cofactor is a non-protein molecule that binds to an enzyme and is required for its biological activity.
What are some common cofactors?
Fe2+
Cu2+
K+
Se
Zn2+
What is a coenzyme?
A nonprotein organic substance that usually contains a vitamin or mineral and combines with a specific protein, the apoenzyme, to form an active enzyme system.
What is a prosthetic group?
The nonprotein component of a protein. For example, the heme group in hemoglobin.
The nonprotein portion of the overall protein is usually covalently bound to the protein.
True or False: Enzymes are specific.
True. Enzymes display a high degree of specificity and selectivetity for their substrate.
What controls the specficity of an enzyme?
The structure of an enzyme controls its specificity.
How may one get a clue to what reaction an enzyme catalyzes?
The enzyme name gives clues as to the reactions that are catalyzed by a particular enzyme.
What are hydrolases?
Hydrolases are enzymes that catalyze hydrolytic cleavage reactions.
What are nucleases?
Nucleases are enzymes that break down nucleic acids.
What are proteases?
Proteases are enzymes that break down proteins.
What are synthases?
Synthases are enzymes that are used in anabolic reactions that polymerize two smaller molecules.
What are polymerases?
Polymerases are enzymes that catalyze the polymerization of nucleic acids.
What are kinases?
Kinases are enzymes that catalyze the addition of a phosphate group to a molecule.
Protein kinases add a PO4 to a protein.
What are phosphatases?
Phosphatases catalyze the removal of a phosphate group.
What are ATPases?
ATPases are enzymes that hydrolyze ATP.
What are the six major classes of enzymes and what do they catalyze?
1) Oxidoreductases - Oxidation & Reduction rxn’s
2) Transferases - Group transfer
3) Hydrolases - Hydrolysis rxn’s (Transfers functional groups to water)
4) Lyases - Addition or removal of groups to form double bonds
5) Isomerases - Isomerization (intramolecular group transfer)
6) Ligases - Ligation of two substrates at the expense of ATP hydrolysis
What is a nucleophile?
Nucleophiles are molecules that have relatively rich pi bonds or lone pairs that act as electron sources in the making of new bonds.
What is an electrophile?
Electrophiles are molecules with relatively electron poor atoms and they serve as acceptors (or sinks) for electrons from nucleophiles.
What are examples of nucleophiles?
What are some common enzymatic reactions?
1) Make a bond between a nucleophile and an electrophile.
2) Break a bond so that stable molecules/ions are created
3) Add protons - The molecule has a strongly basic functional group or there is a strong acid present
4) Take a proton away - The molecule has a strongly acidic proton (amino acids) or there is a strong base present.
How do enzymes work?
Enzymes work by lowering the activation enegry (Kinetic barrier) that a molecule needs to “become” a product.
Before a substarte can become a product, it must have a certain minimum energy to pass through a transition state.
What is the significance of the transition state in enzyme catalyzed reactions?
When bonds are made/broken, charged intermediates are often formed which have a higher energy than the reactants.
The transition state is even higher in energy and may resemble the charged intermediate.
The goal, therefore, is to stabilize the charges and hence lower the energy of the transition state.
Picture of transition state in enzyme catalyzed reactions.
What is the ‘Lock and Key’ hypothesis of enzyme function?
The enzyme is the lock and the substrate is the key.
What is the ‘Induced Fit’ hypothesis?
When a substarte binds an enzyme, a conformational change occurs in the enzyme.
This is the model that is prevalent in most enzyme-substrate interactions.
Non-covalent bonds stabilize the interactions between the enzyme and substrate.
True or False: Enzymes are most complementary to substrate in the transition state rather than the ground state.
True; The primary job of the enzyme is to maximize favorable interactions with the transition state, NOT the starting substrates.
Enzymes bind the transition state more tightly than it binds the original substrates or products.
True or False: Stabilizing the transition state results in enhancing the rate of catalysis.
True: After bond breaking/making at the transition state, the products are weakly bound by the enzyme. This results in loss of the product.
As bonds are broken/made, changes in charge may occur.
The enzyme can bind the transition state more tightly than the substarte because of the stabilization provided by direct interactions between the enzyme and transition state.
What provides evidence of the transition state theory?
The tight binding of transition state analogs is evidence of the transition state theory.
Transition state analogs are stable molecules that are chemically / structurally similar to the transition state substrate.
How does an enzyme invoke catalysis and accelerate the rate of reaction?
1) Stabilize transition state - Via electrostatic stabilization
2) Destabilize enzyme-substrate complex
3) destabilize intermediates
How exactly does the stabilization of the transition state occur in enzyme catalysis?
Enzymes stabilize the transition state by the use of acid / base catalysis.
The catalysis is done by either Hydronim (H3O) or Hydroxide (OH- ).
Removing a proton decreases the developing of a positive charge.
What are some of the important amino acid groups that are involved in stabilizing the transition state?
Asp
Glu
His
Cys
Tyr
Lys
True or False: Enzymes can use acid and base catalysis simultaneously.
True
True or False: The pKa values of an amino acid can be different in an amino acid side chain at the active site of an enzyme from those in solution.
True
How do metal ions influence enzyme catalysis?
Metal ions can act as electrophiles and stabilize/shield the increased electron density and negative charge.
Metals can also be a nucleophile at neutral pH.
Metals can oreint the substrate and stabilize the charge on the substrate.
What is the relationship of covalent or nucleophilic catalysis in enzyme-substrate reactions?
There is a transient formation of an enzyme-substrate covalent bond.
The nucleophilic group on the enzyme donates a proton to the electrophilic group on the substrate.
The original nucleophile can ten interact with the intermediate in a nucleophilic substitution rxn
The nucleophile catalyst and the original nucleophile interact w/ a carbonyl carbon in a substitution rxn and form a tetrahedral oxyanion intermediate
What makes a good covalent catalyst and what are some examples?
A good covalent catalyst must be highly nucleophilic and form good leaving groups.
Examples of these would be imidazole and thiols
Lys
His
Cys
TPP
Picture of Imidazlole ring.
Picture of a thiol group.
What does enzyme catalysis and proximity, orientation and strain have to do with one another?
Enzymes bring substrates and catalytic groups together
Enzymes orient these groups for catalysis
Enzymes facilitate attaining the transition state
Uses intermolecular and intramolecular strain to achieve this; Intramolecualr strain is faster at achieving catalysis
How does destabilization of the enzyme-substrate complex and catalysis go hand-in-hand?
When the enzyme and substrate come together, entropy is lost
This reduces the translational and rotationaly mobility of the substrate
Desolvation makes some groups in the enzyme substrate complex more reactive by
a) Substrate has lower dielectric constant environment
b) Exposes H2O bonded groups for electrostatic catalysis
c) Raises energy of the enzyme-substrate complex and makes it nmore reactive
How can destabilization of the enzyme-substrate complex by electrostatic charges increase the rate of reaction?
Electrostatic destabilization of a substrate may result in a rate increase
This occurs because of the juxtaposition of like charges in the active site
This leads to charge repulsion that may be relieved in the course of a reaction
What are enzyme kinetics?
Enzyme kinetics deals with factors that affect the rate of enzyme catalyzed reactions.
Enzyme concentration
pH
Ionic strength
Temperature
Ligand concentration
What is the Michalis-Menten equation?
Km is the concentration of substrate that leads to half-maximal velocity. Km = 1/2 Vmax
Vmax is the limiting velocity as substrate concentrations get very large. Vmax (and V) are expressed in units of product formed per time.
If you know the molar concentration of enzyme, you can divide the observed velocity by the concentration of enzyme sites in the assay, and express Vmax as units of moles of product formed per second per mole of enzyme sites.
What is Km?
Km is expressed in units of concentration, usually in Molar units.
Km is the concentration of substrate that leads to half-maximal velocity. Km = 1/2 Vmax
Small / low Km values mean tight binding of the enzyme to substrate
Large / high Km means weak binding of the enzyme and substrate
Km is a measure of how well an enzyme binds to a substrate
How does concentration and Km go hand in hand?
If an enzyme is present and the substrate concentration is below the enzymes km, most of the enzymes binding sites will be free
If an enzyme is present and the substrate concentration equals the enzymes km, most of the enzyme binding sites will be taken
If an enzyme is present and the substrate concentration is above the enzymes km, then all of the enzyme binding sites will be taken up
What is Vmax?
Vmax is the maximum velocity or rate at which the enzyme catalyzed a reaction. It happens when all enzyme active sites are saturated with substrate.
Since the maximum velocity is described to be directly proportional to enzyme concentration, it can therefore be used to estimate enzyme concentration.
Vmax is never achieved as it is a theoretical maximal rate
Vmax is constant
To reach Vmax, all enzyme must be tightly bound to substrate
Vmax is asymptotically approached as substrate is increased
What is turnover?
Turnover is measured by Kcat
It is a measure of catalytic activity of the enzyme
It specifically measures the number of substrate molecules converted to product per enzyme molecule per unit time when ezyme is saturated with substrate.
If the Michiles-Menten model is true, then K2 = Kcat = Vmax / Et
What is the catalytic efficiency of an enzyme?
This is Kcat / Km
It is an estimate of ‘how perfect’ an enzyme is
This is a second rate-order constant
Measures how an enzyme functions when substrate concentration is low
The upper limit for Kcat / Km (108 to 109 M1 sec1 ) is the diffusion limit or the rate at which enzyme and substrate diffuse together
Picture of Lineweaver-Burke equation.
What are some ways that enzyme activity can be regulated?
10 Amount of substrate
2) Gene regulation
3) Regulatory molecules / Modulator proteins
4) Competition for active site
5) Allosteric regulation
6) Feedback control (Positive and negative)
7) Covalent modification
8) Phosphorylation / dephosphorylation
9) Cleavage (zymogens)
10) Isozymes - Enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters (e.g. different KM values), or different regulatory properties.
What factors effect the availability of an enzyme?
Compartmentalization
Transcription / translation
Protein processing / protein degredation
Graph showing the effects of inhibition on an enzyme.
Graph showing Michelis-Menten kinetics.
Picture showing allosteric enzyme regulation.
Picture showing allosteric negative feedback inhibition of an enzyme.
Picture of enzyme positive feedback.
Picture showing different modes of inhibition of an enzyme.
What are the classes of reversible enzyme inhibitors?
a) Competitive inhibitors - Bind only the enzyme. Compete with substrate for enzyme binding site. Resemble the structure of the substrate. Lead to changes in Km but NOT Vmax. If substrate concentration is high enough, this can be overcome. Function as drugs. (See slide about Malonate inhibition of Succinate)
b) Uncompetitive inhibitors - This type of inhibitor binds to the enzyme-substrate complex. Keeps catalysis from happening by not allowing product to form. Change Vmax but not Km.
c) Noncompetitive / Mixed inhibition - Inhibitor binds to either the enzyme and/ o r the enzyme-substrate complex. It may or may not change substrate binding affinity. This alters the binding pocket of the enzyme. This is hypothetical.
Graph of Michelis-Menten Non-competitive inhibition.
Graph of Mechelis-Menten competive and non-competitive inhibition.
Graphs of competitive inhibitors.
Km changes; Vmax does not change.
Graph of uncompetitive inhibition.
Km and Vmax both change.
Different types of inhibition.
NOTE: In mixed inhibition Km and Vmax both change.
How does enzyme inhibition relate to metabolism?
Many end products are inhibitors of key steps in catalysis.
Drugs can be designed to inhbit key steps in a metabolic pathway.
Irreversible / covalent inhibition is accomplished using certain compounds. (Inhibit transition state)
What is allosteric enzyme regulation?
Allosteric regulators bind at a site on the enzyme other than the active site
Enzymes that are situated at key steps in a metabolic pathway are modulated by allosteric regulators
These regulators are usually made elsewhere in a pathway
They may regulate by positive or negative feedback
Kinetics of allostericly regulated enzymes are sigmoidal )S-shaped)
This type of regulation leads to changes in concentration of enzyme and therefore change Vmax but not Km
Picture of chymotrypsin binding site.
Chymotrypsin mechanism of catalysis.
Describe the chemistry of the active site of an enzyme.
The active site acts as a flexible structure that binds substrate and initiates the conversion to the transition state
By stabilizing the transition state, the enzyme greatly increases the concentration of the reactive intermediate that can be converted to product
What are factors that affect reaction velocity?
Substrate concentration
- Maximal velocity: The rate or velocity of a reaction (V) is the number of substrate molecules converted to product per unit time. The rate of an enzyme catalyzed rxn increases w/ substrate conc. until Vmax is reached.
- Shape of enzyme kinetics curves: Most enzymes show Michaelis-Menten kinetics which plots initial rxn velocity Vo against substrate conc. The curve is usually hyperbolic. Allosteric enzymes, however, show sigmoidal binding kinetics.
Temperature
- Increase of velocity with temperature: The reaction velocity increases with temperature until a peak velocity is reached.
- Decrease of velocity with higher temperature: Eurther elevating temperature causes a decrease in rxn velocity because of temperature-induced denaturation of the enzyme.
pH
- Ionization of active site: The concentration of H+ affects rxn velocity. The enzyme and substrate must have specific chemical groups in either an ionized or unionized state to be able to react.
- Denaturation: Extreme pH’s can denature enzymes
What does Vmax mean?
What does Km mean?
Km is the substrate concentration that gives half maximal velocity of an enzymatic reaction.
Measure of the affinity of enzyme and substrate with the higher the KM, the lower the affinity.
Called also Michaelis constant; Michaelis-Menten constant.
What are some characterisctics of Km?
Km does not vary with enzyme concentration
Small Km leads to a high binding affinity for substrate because a low concentration of substrate is required to half saturate the enzyme, that is, reach a velocity of 1/2 Vmax
Large Km lead to low affinity for substrate because a high conc. of substrate is needed to reach 1/2 Vmax
What is the relationship of velocity to enzyme concentration?
The rate of reaction is directly proportional to the enzyme concentration at all substrate concentrations.
For example, if the enzyme conc. is halved, the initial rate of reaction (Vo ) as well as Vmax are reduced to half that of the original.
How does competitive inhibition effect Vmax and Km ?
The effect of a competitive inhibitor on Vmax is reversed by incresing substrate conc.
Competitive inhibition increases the apparent Km . This means that in the presence of a competitive inhibitor, more substrate is needed to achieve 1/2 Vmax
How do non-competitive inhibitors effect Vmax and Km ?
Non-competitive inhibitors decrease apparent Vmax as it cannot be overcome by increasing substrate conc.
Non-competitive inhibitors do not interfere with the binding of substrate to enzyme. As such, they have no effect on Km .
What is an isozyme?
These are enzymes that catalyze the same reactions but do not have the same physical properties because of their different amino acid sequences.
What are the traits of an allosteric enzyme?
a) Composed of multiple subunits
b) Catalyze the committed step
c) Bind substrate cooperatively
d) Show a sigmoid binding curve whne Vo is plotted against [S]
What are the mechanisms of enzyme catalysis?
Substrate proximity and orientation
Acid / base catalysis
Describe the reaction mechanism of chymotrypsin.
Chymotrypsin uses acid / base catalysis
There is a serine at the active site that acts as a base / proton acceptor during the hydrolysis of a peptide bond
Step I: The serine in the enzymes active site attacks the peptide bond to be cleaved
Step II: A covalent complex is formed between the enzyme and the portion of the substrate that is to be cleaved
Step III: The enzyme - substrate complex is hydrolyzed which releases the peptide
