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Enzymes are catalysts that allow biological reactions to occur at a faster rate. Common names for enzymes are usually formed by adding the suffix -ase to the name of the reactant. A true catalyst increases the rate of reaction by lowering the activation energy barrier.
Enzymes are catalysts that allow biological reactions to occur at a faster rate. Common names for enzymes are usually formed by adding the suffix -ase to the name of the reactant. A true catalyst increases the rate of reaction by lowering the activation energy barrier.
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Enzymes are catalysts that allow biological reactions to occur at a faster rate. Common names for enzymes are usually formed by adding the suffix -ase to the name of the reactant. A true catalyst increases the rate of reaction by lowering the activation energy barrier.
Copyright:
Attribution Non-Commercial (BY-NC)
Formati disponibili
Scarica in formato PDF, TXT o leggi online su Scribd
Enzymes catalysts that allow biological reactions to
occur at a faster rate
Ea energy input required to initiate the reaction Properties of A True Catalyst Increases the rate of reaction by lowering the activation energy barrier
Not used up or permanently changed during the catalytic process
Does not change the position of equilibrium, only the rate at which equilibrium is attained
Usually acts by forming a transient complex with the reactant, thus stabilizing the transition state Enzyme Cofactor may be an organic or organometallic molecule (coenzyme) or a metal ion ( Zn 2+ , Mg 2+ ,
Cu 2+ )
Holoenzyme complete molecular package, protein and cofactor
Apoenzyme protein component
Naming Enzyme Common names for enzymes are usually formed by adding the suffix ase to the name of the reactant Tyrosinase catalyzes the oxidation of tyrosine Cellulase catalyzes the hydrolysis of cellulose to produce glucose
Some early enzyme names are less descriptive and give no clue of their function or substrates Trypsin, chymotrypsin Enzyme nomenclature 6 major classes, each w/ subsclasses according to rxn catalyzed
Each is assigned with a 4-digit number and systematic name which identifies the rxn catalyzed.
Enzyme nomenclature 4-digit official number 1 st digit: class name 2 nd digit: subclass 3 rd digit: accepting functional group 4 th digit: accepting molecule
EC 1 Oxidoreductases EC 1.1 Acting on CH-OH group of donors EC 1.2 Acting on aldehyde or oxo group of donors EC 1.3 Acting on CH-CH group of donors EC 1.4 Acting on CH-NH2 group of donors EC 1.5 Acting on CH-NH group of donors EC 1.6 Acting on NADH or NADPH
Kinetics In a reaction of the form A + B P, the rate can be expressed in terms of the rate of disappearance of one of the reactants or in terms of the rate of appearance of the product
Rate of disappearance of A = -A[A]/At where A symbolizes the change on [A] and t is time
Rate of appearance of P = A[P]/At So we can express rate in terms of any of these : Rate = -A[A]/At = -A[B]/At = A[P]/At Kinetics It is established that Rate o [A] f [B] g
Rate = k [A] f [B] g
Where k is the rate constant (proportionality factor that accounts for how well the reacting molecules oriented during collisions)
Exponents f and g usually small whole numbers 1 or 2 or in some cases 0 must be determined experimentally The overall order of the reaction is the sum of all the exponents
Rate = k[A] 1 first order reaction w/ respect to A and first order overall
Kinetics First order reaction rate depends only on one reactant
Second order reaction the rate of reaction depends on the two reactants Rate = k[A] 1 [B] 1 first order wrt A and first order wrt B and second order overall
Zero order reaction rate is constant depends not on concentrations of reactants but on other factors (presence of catalyst) A B, Rate = k[A] 0
Kinetic Properties of Enzymes Initial rate (u 0 ) determined during the first few minutes of the reaction Maximum velocity (V max )
Michaelis-Menten Equation Leonor Michaelis and Maud Menten explain the hyperbolic rate curve Proposed that enzyme molecules, E, and substrate molecules, S, combine in a fast and reversible step to form an ES complex E + S k1 k2 ES k3 k4 E + P where k are rate constants
Assumptions: 1. Neglect the reaction that reverts product P and free enzyme to the ES complex defined by k4 2. ES complex is a steady-state intermediate After mixing E and S, a certain level of ES is formed and its concentration remain constant because it is produced at the same rate as it breaks down
Time course of S consumption, P formation and establishment of steady state level of ES
Bottom curve: detail of early stage of time course M-M equation ] [ ] [ max 0 S K S V M + = v Km Km Michaelis constant In E + S k1 k2 ES k3 E + P, Km is expressed as:
Let Km = [S]
] [ ] [ max 0 S K S V m + = v ] [ ] [ ] [ max 0 S S S V + = v ] [ 2 ] [ max 0 S S V = v 2 max 0 V = v 1 3 2 k k k Km + = Km Remember:
Km becomes:
This is equal to the equilibrium constant for the dissociation of the ES complex ES k2 k1 E + S
So when k2>>k3, Km is simply the dissociation constant for the ES complex
1 3 2 k k k Km + = 1 2 k k Km = 1 2 ] [ ] ][ [ k k ES S E Keq = = Km Km measure of how tightly the substrate is bound to the enzyme
The greater the value of Km, the higher is k2 than k1
Km is the inverse measure of the affinity of the enzyme for the substrate
Vmax and K3 If the substrate concentration is so high so that E is completely saturated : [ES] = [E T ] and V= Vmax
From this part of the enzyme equation: ES k3 k4 E + P
Turnover number, k3 (kcat or kp) number of moles of substrate transformed to product per mole of enzyme in a defined time period ] [ max 3 T E V k = ] [ 3 ES k V = ] [ 3 max T E k V = K M , K cat Carbonic anhydrase -CO 2 : K cat = 1 x 10 6 s -1 ; K M = 1.2 x 10 -2 M Carbonic anhydrase -HCO 3 - : K cat = 4 x 10 5 s -1 K M = 2.6 x 10 -2 M
Which is better substrate for the enzyme? K M , K cat Carbonic anhydrase - CO 2 : K cat = 1 x 10 6 s -1 ; K M = 1.2 x 10 -2 M
Carbonic anhydrase - HCO 3 -
K cat = 4 x 10 5 s -1 K M = 2.6 x 10 -2 M
K cat /K M = 8.3 X10 7 M -1 s -1 K cat /K M = 1.5 X10 7 M -1 s -1 Lineweaver-Burk Equation Double reciprocal plot allows one to plot experimental enzyme rate data in the form of a straight line
y = m x + b ] [ ] [ 1 max 0 S V S K M + = v max max 0 1 ] [ 1 1 V S V K M + = v ] [ ] [ ] [ 1 max max 0 S V S S V K M + = v ] [ ] [ max 0 S K S V M + = v max max 0 1 ] [ 1 1 V S V K M + = v Other Methods of Km and Vmax Determination Eadie-Hofstee Plot The Michaelis-Menten equation is rearranged to
This is a graph of u 0 vs u 0 /[S] where: Slope = -Km Y-intercept = Vmax X-intercept = Vmax/Km max 0 0 ] [ V S Km + = v v Eadie-Hofstee Other Methods of Km and Vmax Determination Hanes-Woolf Plot The Michaelis-Menten equation is rearranged to
This is a graph of [S]/u 0 vs [S] where: Slope = 1/Vmax Y-intercept = Km/Vmax X-intercept = -Km max max 0 ] [ 1 ] [ V Km S V S + = v Hanes-Woolf Characteristics of Enzyme Reactions 1. Enzyme
2. pH enzymes have an optimal pH at which they function most effectively (pH 6-8) Characteristics of Enzyme Reactions 1. Enzyme
2. pH enzymes have an optimal pH at which they function most effectively (pH 6-8)
3. Temperature enzymes are sensitive to temperature changes
For most enzymes, the rate decline begins in the temp. range of 50C to 60 C Binding of Substrate to Enzyme Active site specific region in the enzyme where the substrate specifically binds
Pocket or crevice in the 3-D structure of the enzyme Consists of certain amino acids that may be involved with the noncovalent interaction with the substrate Characteristics of The Active Site 1. Specificity it is able to discriminate among possible substrate molecules Two Types 1. Absolute accept only one type of molecule (can even discriminate between a D or L isomer)
2. Group Specificity accept a number of closely related substances as long as the reactive functional group is present Characteristics of The Active Site 2. Relatively small, 3-D region within the enzyme aa residues need not be contiguous in the linear protein chain
3. Holds substrate through weak, noncovalent, reversible interactions hydorphobic, ionic and H- bonding First Step Binding of substrate to the enzyme Three Models Lock and key model
Induced-fit model
Transition-state model Lock-and-Key model Induce-fit model Transition state model Increase in Reaction Rate
1. Entropy loss in the ES formation 2. Destabilization of ES due to strain, desolvation or electrostatic effect Loss of Entropy AG = AH - TAS
ES complex is highly organized compared to E and S in solution
E and S have translational entropy (freedom to move in 3-D) as well as rotational entropy (freedom to rotate or tumble about in an axis)
Destabilization of ES Complex By strain or distortion consequence of the fact that the enzyme is designed to bind the transition state more strongly than the substrate
By desolvation of charged groups in the substrate charged groups are highly stabilized in water
By electrostatic destabilization when a substrate enters the active site, charged groups may be forced to interact with groups with same charge resulting to repulsion and destabilization 2 nd Step The transition state is formed and catalysis can occur
Proximity and orientation speed up the reaction in the transition state, the substrate is bound close to atoms with which it is to react and also placed in the correct orientation wrt those atoms Mechanistic Features of Enzymes General Acid-Base Catalysis Step 1 H + is added to the carbonyl group Step 2 formation of the tetrahedral intermediate Step 3 a proton is transferred from O to N Step 4 a base will assist by accepting the proton from the intermediate Mechanistic Features of Enzymes Metal-ion Catalysis alkali metal ions (Na+, K+) and transition metals (Mg 2+, Mn 2+, Cu 2+ , Zn 2+ ,
Fe 2+ ,
Fe 3+ ,
Ni 2+ ,
and others)
1. Holds a substrate properly oriented by coordinate covalent bonds Mechanistic Features of Enzymes Metal-ion Catalysis
2. Enhance reaction by polarizing the scissile bond or by stabilizing a negatively charged intermediate Mechanistic Features of Enzymes Metal-ion Catalysis
3. Participate in biological oxidation-reduction reactions by reversible electron transfer between metal ions and substrate
Acts as a Lewis acid by accepting electrons Mechanistic Features of Enzymes Covalent Catalysis nucleophilic substitution reaction A nucleophilic, (electron-rich) functional group attacks an electron-deficient group Nucleophile Leaving group Active Site Events Functional groups in the active site that can have catalytic roles:
Imidazole ring of histidine Hydorxyl group of serine Carboxyl side chain of aspartate and glutamate Sulfhydryl group of cysteine Amine group of lysine Phenol group of tyrosine
Mechanism of Chymotrypsin Action Serine residue at position 195 is required for activity Another critical aa in chymotrypsin is His 57 Ser 195 His 57 Asp 102 Enzyme Activity Regulation Irreversible inhibitor forms covalent or very strong noncovalent interactions with the enzyme E-H + R-X E-R + HX (active) (inactive)
Example: aspirin (acetylsalicylic acid) Acts by blocking synthesis of pain-producing prostaglandins Reversible Inhibitors Can readily combine with and dissociate from an enzyme and render the enzyme inactive only when bound
EI is held by weak, noncovalent interactions similar to ES complex Competitive Inhibition Inhibitor usually resembles the structure of normal substrate and is capable of binding to the active site of the enzyme
Transition state analogs modeled after the structures of substrate in presumed transition states
Noncompetitive Inhibition Inhibitor can bind to the enzyme at a site other than the active site of the enzyme Binding of inhibitor causes a change in the structure of the enzyme especially around the active site Uncompetitive Inhibition Inhibitor binds only to the ES complex but not with the free enzyme Influence the activity of the enzyme only when [S] is high and, in turn, the ES concentrations are high Kinetics of Inhibition Competitive inhibition slope and x-intercept of Lineweaver-Burk plot change but the y-intercept does not Km increase more substrate is needed to get the velocity of half the maximum velocity Kinetics of Inhibition Pure noncompetitive inhibition - slope and y- intercept of Lineweaver-Burk plot change but the x- intercept does not Pure - Vmax decreases but Km remains the same because the inhibitor does not interfere with the binding of substrate to the active site
Mixed Vmax decreases and Km increase because the inhibitor affects the binding of the substrate to the active site
Kinetics of Inhibition Uncompetitive inhibition Vmax and Km both decrease
Reversal of inhibition is not achieved by increase in [S] Allosteric Enzymes
Concentration of final products (feedback inhibition) Concentration of the beginning substrate Concentration of an intermediate formed in the sequence Concentration of external factors (hormones)
Effectors Biomolecules that influence the action of an allosteric enzyme Positive effectors stimulants Negative effectors - inhibitors Catalytic and Regulatory Sites Catalytic site where substrate binds
Regulatory site the binding of effector molecules changes the conformation of the protein in a way that tells the other subunits that it is bound Chemical Alterations 1. Phosphorylation of hydroxyl groups of serine, threonine or tyrosine
2. Attachment of an adenosyl monophosphate to a hydroxyl group
3. Reduction of cysteine disulfide bonds Catalyzed by phosphorylase kinase Catalyzed by phosphorylase kinase Catalyzed by phosphorylase phosphatase Glutamine synthetase Proteolytic Cleavage Zymogen inactive protein precursor Cleaved at one or a few specific peptide bonds to produce the active form of the enzyme Regulation by Isoenzyme Isoenzymes multiple forms of enzymes that have similar but not identical amino acid sequences
May demonstrate the same enzyme activity but the may differ in kinetics (Km and Vmax), differ in effectors, differ in the form of coenzyme needed and cellular distribution