Chpt 8 Flashcards
Enzyme Def
a protein or RNA molecule that catalyzes biochemical reactions
Common name convention
substrate name+ rxn performed+ -ase suffix
Exceptions
Trypsin and Chymotrypson
7 Classes of enzymes
OTH LIL-T; 2-CNP; 4-CNS; 6CONS
EC1-oxidoreductase- catalyzes oxidation/reduction reactions (Dehydrogenase)
EC2-transferase- catalyzes the transfer of C, N, or P containing groups (kinase)
EC3- Hydrolase- catalyzes the cleavage off bonds by addition of water
EC4-Lyase-catalyzes the cleavage of C-C, C-S, and some C-N bonds
EC5-Isomerase- catalyzes the racemization of optical and geometric isomers
EC6-Ligase- catalyzes the formation of bonds to C, O, N, and S coupled to hydrolysis of a High Energy Phosphate
EC7-translocase- catalyzes the moment of a molecules from side 1 of a membrane to side 2 (the other side) of the membrane
Common names for Metabolic Enzymes
KIM-DA; TiiO_
Kinase-(transferase)-transfer a phosphate from one molecule (ATP) to another molecule
Isomerase (isomerase)
-converts to another isomer
-changes atom configuration without losing or gaining atoms
Mutase (isomerase)-shifts a phosphate from one carbon to another carbon within the SAME molecule
Dehydrogenase (Oxidoreductase)-involves NADH and FADH; oxidation/reduction reaction
Aldolase-cleves C-C bonds (Reverse Aldol condensation)
Properties of Enzymes (5)
1) Active site
- 3D pocket or cleft created by catalytic groups which are amino acids who R groups interact to cause the protein to fold into tertiary structure forming the active site
- specific for particular substrate/reaction
2) Enzyme “Helper” Molecules
- cofactor-inorganic metal ion; dissociable from enzyme: ex: Mg2+ and Fe2+
- coenzyme-organic molecules; dissociable from enzyme: ex: NADH, FADH, NADHP and oxidized forms
- prosthetic group-organic molecules that is covalently bonded to enzyme (can’t dissociate) Ex: Heme
* *prosthetic groups and coenzymes are derivatives of Vitamins
3) Catalytic Efficiency
- increases rate of reaction compared to uncatalyzed
- 100 to 1000 substrates convert to products/sec
- turnover-# of substrate molecules/#of enzymes per sec
4) Regulation- enzymes can be activated or inactivated
5) Compartmentalization- enzymes are localized within particular compartments of a cell
Apoenzyme vs holoenzyme
Apoenzyme- Enzyme without helper molecule
Holoenzyme-enzyme with helper molecule
Thermodynamics
Total E= Usuable E + Unusable E
H=G+TS–>convert to deltaG=deltaH-TdeltaS
Gibbs Free Energy (G)
Negative Delta G
-spontaneous
substrate A is at a higher E than substrate B, thus substrate A must release E-EXERGONIC-release E
Positive Delta G
-nonspontanous, but spontaneous in reverse reaction
substrate A is at lower E than substrate B; thus has to consume energy to go to Higher E-ENDERGONIC-energy consumed
Zero Delta G; equilibrium and any living system equilibrium means death
Considerations of Gibbs free Energy
- tells us nothing about the rate of rxn instead tells us about spontaneity
- deltaG=the difference between Reactants E and Products E
- deltaG=is independent of the path of the reaction
Enzymes
- equilibrium
- reaction rate
Alter Rate but NOT EQUILIBRIUM
1) Enzymes do not change equilibrium of reaction
- amount of product formed is the same despite presence/absence of enzyme
2) Rate is Significantly different between a enzyme catalyzed reaction and uncatalyzed reaction to reach equilibrium
3) *** Enzymes accelerate the attainment of equilibrium, BUT DO NOT shift their position. The equilibrium position is a function only of the free energy difference between products and reactants
Enzymes accelerate reactions by lowering activation E (G=) by facilitating the formation of transition state
Evidence of ES complex
1) Saturation of active site by increasing substrate concentration [S]
- at constant enzyme concentration [E], the velocity increases with increasing substrate concentration [S] until a maximum velocity is reached.
- at maximal velocity all active sites are occupied
- uncatalyzed reactions do not exhibit this effect
2) X-ray crystallography
- X-ray structures show interaction between R groups of amino acids (catalytic group) in the active site and the substrate
3) Spectroscopic Characteristics
- changes in absorbance/fluorescence upon mixing substrate and enzyme
Active Site vs Allosteric Site def
Active site-region of the enzyme that binds the substrates and cofactors (if needed)
Allosteric Site-binding site other than active site
Active site characteristics
1) 3D crevice
- formed by catalytic groups- the amino acids involved in the active site have R groups that span a vast area of the linear protein and interact with one another to allow the protein to form into tertiary structure and forms active site
2) represents a small part of the total volume of enzyme
- the amino acids not involved in the active site serve as scaffolding
3) Creates UNIQUE MICROENVIROMENT
- water is often excluded (unless it participates in the reaction) creating a non polar enviroment
- Polar amino acid R groups often acquire “special” properties
4) binds substrate to the enzyme by multiple weak attractions due to Van Der Waals forces, Hydrogen Bonding, and Hydrophobic interactions NOT Covalent Bonding
- weak and reversible
5) has specificity of binding dependent on the arrange of atoms-2 TYPES
- lock and key model-active site shape matches the shape of substrate
- induced fit model-shape of active site undergoes conformation change as the substrate binds
Binding energy of enzyme
free energy released when the enzyme binds to the substrate
-Most E is released when the transition state is reached
Kinetics and Enzyme kinetics Def
Kinetics-the study of the rate of a chemical rxn
Enzyme Kinetics-study of the rate of enzyme catalyzed rxn
Kinetics:
- What is Rate
- Rxn order
What is Rate?
Rxn= A->P
V= -deltaA/deltaT= deltaP/deltaT
-rate of disappearance of substrate A and rate of appearance of product
Rxn Order
First Order-rates are directly proportional to reactant concentrations
A-> P; V=k[A]
2nd Order-Bimolecular Rxn(takes two molecules)
2A->P; V=k[A]^2 OR A+B-> P; V=k[A][B]
-pseudo first order- where substrate B is in excess conc and substrate A is low conc., the rxn will be first order for
Zero Order
-rate of rxn is independent of reactant concentration
How are reaction rates Studied?
ONLY look at initial velocities (Vo) so no product in the tube!
-Measure increase in concentration of Products over time at different substrate concentrations
Vo=deltaP/deltaT
- where Vo is the number of moles or product formed per second
- approaches equillibrium
Draw a Michaelis Menten Graph with:
- Vmax
- x and y axis labeled
- Km labeled
Hyperbolic curve
X-axis= Substrate Conc [S]
Y axis= Rxn Velocity (Vo)
Vmax/2 on the Y axis and where it lines up on the curve to the x-axis=Km
Km
is a characteristic of an enzyme and its particular substrate
1) Km is the substrate conc [S} to produce 1/2Vmax
- not affected by enzyme conc
2) ratio of rate constants which shows the strength how tightly the substrate is being bounded by the enzyme (STRENGTH OF ES COMPLEX)
- If K-1>K2, ES complex dissociates into E+S faster than E+P
- small Km=high affinity of enzyme to substrate; very little substrate required to saturate enzyme
- High Km=low affinity of enzyme to substrate;Alot of substrate required to saturate enzyme
Vmax
- def
- equation
Number of substrate molecules converted to product by an enzyme molecule in a time where enzyme is saturated
Vmax=k2[E]t–> K2=Vmax/[E]t= K2=Kcat which is the catalytic rate constant
Ethanol Sensitivity
Two forms of aldehyde dehydrogenase (oxidation/red)
- Mitochondrial form-low Km
- Cytoplasmic Form-High Km
Sensitive people have less active mitochondrial enzyme due to an amino acid substitution
- thus acetylaldehyde is process only by cytoplasmic enzymes
- with High Km this enzyme achieves a high rate of catalysis only at high concentration of acetylaldehyde
Symptoms-Facial flushing and tachycardia
Factors effecting Reaction Velocity
Temperature
- velocity increases as temperature increases; more substrate molecules have sufficient E to reach transition state
- decrease of velocity due to denaturation of enzyme
pH
- affects the protonation of the R groups of amino acids involved in catalysis
- Excessive pH denatures Enzymes
- pH optimum varies
Catalytic Efficiency
Property of Enzymes-increased rate of rxn compared to uncatalyzed
catalytic efficiency=Kcat/Km
-allows comparing enzymes preference for different substrates
When [S]»Km the rate=Vmax thus Kcat=Vmax/[E]t
-this situation not typical under physiological conditions
UNDER PHYSIOLOGICAL CONDITIONS: enzyme active sites are not saturated SO:
-When [S]
How efficient can an Enzyme Be?
CONTROLLED BY DIFFUSION
Catalytic efficiency (Kcat/Km) cannot exceed the diffusion- controlled encounter of an enzyme and its substrate; Diffusion can be controlled by confining substrate to multi enzyme complex
- K1, rate constant for formation of ES complex, is limited by diffusion to 10^9 to 10^9/ sM, which is the upper limit of Kcat/Km
- Enzymes approaching this level achieve kinetic perfection
ENZYME with Kinetic Perfection
- every Substrate that encounters an Enzyme produces a product
- Enzyme has Circe forces-which is the attractive forces that entice the substrate to the active site
How can Diffusion be controlled?
by confining substrate to multi enzyme complex
Lineweaver-Burke Plot
Graph characteristics
Double Reciprocal-Linear x-axis= 1/[S] Y axis=1/Vo y=mx+b m=Km/Vmax x-int=1/-Km y-int= 1/Vo
Define Inhibitor
a substance that can diminish the velocity of an enzyme catalyzed reaction
What can inhibit the activity of an enzyme?
- specific small molecules or ions
- Drugs or toxics
What are the types of inhibitors?
Reversible inhibitor
- bind weakly (noncovalently) to enzyme
- dilution of enzyme: Inhibitor Complex results in dissociation of inhibitor and recovery of enzyme activity
1) Competitive inhibitors
2) Uncompetitive inhibitors
3) Noncompetitive inhibitors
Irreversible Inhibitor
- bind tightly (covalently or noncovalently) to enzyme
- inhibitors dissociate slowly
Competitive Inhibitor
- Define
- Graph and effect on Vmax and Km
- Overall effect
Type of Reversible Inhibitor
- Where the inhibitor binds to same site (active site) as substrate
- competes for active site; inhibitor is same shape as substrate
GRAPH Slide 68 (chopstick shape when using)
- Vmax remains the same; reversed by increasing [S]
- Increase Km; more substrate need to reach 1/2Vmax
Overall Effect-diminishes the rate of catalysis by reducing proportion of enzyme molecules bound to a substrate
Drug Example of Competitive Inhibitor
Statin Drugs
- antihyperlipidimic agents; atorvastatin(Lipitor) and simvastatin(Zocor)
- completely inhibit the first committed step in cholesterol synthesis (HMG- CoA reductase)
- structural analogs of substrate-inhibits de novo cholesterol synthesis
Uncompetitive Inhibitor
- Define
- Graph and effect on Vmax and Km
- Overall effect
Type of Reversible Inhibitor
- inhibitor binds to ES complex
- substrate has to bind to active site first to form inhibitor binding site where the Inhibitor Binds
Graph SLIDE 71 (two parallel Lines)
- Vmax decreases; Vmax cannot be attained; and cannot be overcome by addition of more substrate
- Km decreases; as [I] Increases
Noncompetitive Inhibitor
- Define
- Graph and effect on Vmax and Km
- Overall effect
Type of Reversible Inhibitor
- inhibitor binds to different site that substrate (allosteric site)
- binds to either free E or ES complex
- *Some form covalent bonds with enzymes
1) Pb forms covalent bonds with sulfhydryl groups of cysteine
2) acetylcholinesterase inhibitors-cleaves the neurotransmitter acetylcholine; found in insecticides and nerve gases
Graph Slide 73 (shape of V)
- decreases Vmax; cannot overcome by increase [S]
- same Km with or without inhibitor; does not interfere with binding of the substrate
Many Drugs Inhibit Enzymes
50% of the ten most commonly dispensed drugs in USA act as enzyme inhibitors
1) Penicillin and Amoxicillin
-inhibit bacterial cell wall synthesis
2)Angiotensin-converting enzyme (ACE) inhibitors
-lower BP by blocking the enzyme that cleaves angiotensin I and angiotensin II (potent vasoconstrictor)
Ex:captropril, enalapril, lisinopril
Reactions with Multiple Substrates
Most reactions contain 2 substrates and 2 products
- Bimolecular and can be categorized into:
1) Sequential Reactions
2) Double Displacement reactions
Sequential Reactions
All substrates bind to the enzyme before any product is released (Ternary Complex)
Two Types:
Ordered (lactate dehydrogenase)
Random (Creatine Kinase)
Ordered Sequential Reactions
-coenzyme and substrate bind in specific order (release of products also occurs in specific order)
Ex: Lactate dehydrogenase
Use Cleland diagrams to show
Random Sequential Reactions
-addition/release of substrates and products is random
Ex: Creatine Kinase
Double Displacement
(Ping Pong)
-One or more products are released before all substrates bind enzymes
-substituted enzyme intermediate is hallmark of ping pong reactions
Ex: Aspartate Aminotransferase
Allosteric Enzymes
-Enzymes that do not follow Michaelis Menten Kinetics
-contain multiple substrate/multiple active sites
-Sigmoidal Plot
-Cooperativity
-Regulatory Enzymes
Ex: Hemoglobin
Regulation of Enzyme activity During Metabolism
Velocity (Vo) of enzymes are responsible to changes in [S]because the intracellular level of many substrates is in the range of the Km
Therefore an increase [S] leads to an increase in Velocity (rate)of the reaction
Allosteric Enzymes may be inhibited or stimulated by binding an effector (also called modifier) which can do either or both:
- Modify the maximal catalytic activity (Vmax) of the enzyme
- alter the affinity of the enzyme for its substrate (Km)
Allosteric Enzymes are often regulated by feedback inhibition
- example of heterotrophic effector-effector is different from substrate
- example of homographic effector-effector molecule is the substrate
