chapter 6 Flashcards
Enzymes: What and Why?
Enzymes are
Most enzymes are
Regulatory enzymes help to
Enzymes function in
Many disease conditions can be related to
Enzymes are catalysts
Increase reaction rates without being used up
Most enzymes are globular proteins
-However, some RNA (ribozymes and ribosomal RNA) also catalyze reactions
Enzymes function in aqueous systems (mild condition: 37°C and pH ≈7)
Regulatory enzymes help to coordinate metabolic pathways
Many disease conditions can be related to inappropriate levels of enzymatic activity
Catalysis has high degree of
-MW:
-Some require additional chemical components for
and they are:
-Holoenzyme/apoenzyme or apoprotein
-Catalysis: high degree of specificity (3D conformation)
-MW: 12 kD to 1,000 kD
-Some require additional chemical components for activity – cofactor (one or more inorganic ions; Coenzyme)
-Coenzyme – complex organic or metalloorganic molecule acting as a functional group carrier
-Prosthetic group – a coenzyme or metal ion that is very tightly or covalently bound to the protein
-Holoenzyme-protein +cofactor in active form
apoenzyme or apoprotein- protein without cofactor
Why biocatalysis over inorganic catalysts?
-Greater reaction specificity: avoids side products
-Milder reaction conditions: conducive to conditions in cells
-Higher reaction rates: in a biologically useful timeframe
-Capacity for regulation: control of biological pathways
Enzyme Classification: 7 classes
- Oxidoreductases- oxi reduces
- Transferases-transfers
- Hydrolases-hydrolysis
- lyases-cleave
- Isomerases-transfer groups in isometric forms
- Ligases-glue
- Translocases-translocate 1 molecule across a membrane
How Enzymes Work: Enzyme-Substrate Complex
active site: the binding pocket on the enzyme where the enzyme-catalyzed reaction takes place
Substrate: the mol. bound in the active site and acted by the enzyme
Binding of a substrate to an enzyme at the active site. The enzyme chymotrypsin with bound substrate (PDB ID 7GCH) (Key aa: red).
∆G‡:
∆G’°
∆G‡: the activation energies
∆G’°: the overall standard free-energy change (S P)
ES and EP:
Enzymes affect
ES and EP: transient complexes
Enzymes affect reaction rates, NOT equilibria, lower activation energy, small ∆G
Reaction rates
S → P
V = k[S] (first order reaction)
k is a proportionality constant reflecting the reaction probability given the reaction conditions
k has units of reciprocal time (s-1)
-1 substrate
V = k[S1][S2] (second order reaction)
k has units of M-1 s-1
-2 substrates, more complicated
Enzymatic Catalysis
-Enzymes do not affect
-Slow reactions face significant
-Enzymes increase
So a lower activation energy means a
-Enzymes do not affect equilibrium (ΔG)
-Slow reactions face significant activation barriers (ΔG‡) that must be surmounted during the reaction
-Enzymes increase reaction rates (k) by decreasing ΔG‡
-So a lower activation energy means a faster rate
Catalytic power: “Lock and Key” Model
Complementary shapes of a substrate and its binding site on an enzyme. The enzyme dihydrofolate reductase with its substrate NADP+, unbound and bound; another bound substrate, tetrahydrofolate, (PDB ID 1RA2). The NADP+binds to a pocket that is complementary to it in shape and ionic properties, an illustration of Emil Fischer’s “lock and key” hypothesis of enzyme action.
ΔGB
ΔGB: Weak binding interactions between the enzyme and the substrate provide a substantial driving force for enzymatic catalysis
binding energy
is the difference between the free energy that is catalyzed and not catalyzed-released by weak interactions
ex: hydrophobic
Catalytic Mechanisms
-acid-base catalysis: give and take protons
-covalent catalysis: change reaction paths
-metal ion catalysis: use redox cofactors, pKa shifters
-electrostatic catalysis: preferential interactions with TS
General acid-base catalysis
-Protons can be transferred to or from a
-Specific acid-base catalytic reactions use only the
-General acid-base catalysts use enzyme
Substrate and proton donors and proton acceptors are located
-Protons can be transferred to or from a charge species that favor breakdown to products instead of reactants
-Specific acid-base catalytic reactions use only the H3O+ or OH- ions present in water
-General acid-base catalysts use enzyme functional groups to assist in proton transfer reactions and facilitate bond cleavage
-Substrate and proton donors and proton acceptors are located side by side
Covalent Catalysis
-A transient covalent bond between the enzyme and the substrate
-Changes the reaction Pathway
-Uncatalyzed: A—B —(H20)–> A+B
-Catalyzed: A—B+X:—>A—X+B—(H2O)-> A+X:+B
Requires a nucleophile on the enzyme
Can be a reactive serine, thiolate, amine, or carboxylate
A transient covalent bond is formed between
A transient covalent bond is formed between enzyme and substrate
Metal Ion Catalysis
-Involves a metal ion bound to the enzyme
-Interacts with substrate to facilitate binding
—-Stabilizes negative charges
-Participates in oxidation reactions
-Nearly a third of all enzymes require a metal ion for activity
large hydrophobic groups bind easily to chymotrypsin. These are the only amino acids that can bind to it. Which are they?
(Phe, and Trp)
Phenylalanine
Tryptophan
Zymogine
inactive form (original form) 1 single chain cut into 3 parts (small pieces) in order to be active and bind to substrate
Role of Serine
functions as a nucleophile because of its hydroxyl group, it donates hydroxyl group to histidine–> (+)
Role of histidine
functions as a general base, accepting a proton from other amino acid, thereby increasing its reactivity,
becomes stabilized by aspartic acid (-)
Role of Asp
its negative charge stabilized the positive charge that develops on Histidine
Steps in chymotrypsin
step 1: substrate binding
step 2:nucleophilic attack
step 3: substrate cleavage
Step 4: water comes in
step 5: water attacks
step 6: break-off from the enzyme
step 7:product dissociates
What is enzyme kinetics?
-Kinetics is the study of the rate at which compounds react
-Rate of enzymatic reaction is affected by:
–enzyme
–substrate
–effectors
–temperature
Why study enzyme kinetics?
-Quantitative description of
-Determine the order of
-Elucidate
-Understand
-Find effective
-Understand regulation of
-Quantitative description of biocatalysis
-Determine the order of binding of substrates
-Elucidate acid-base catalysis
-Understand catalytic mechanism
-Find effective inhibitors
-Understand regulation of activity
How to Do Kinetic Measurements
Experiment:
1.Mix enzyme + substrate
2.Record rate of substrate disappearance/product formation as a function of time (the velocity (V) of reaction)
3.Plot initial velocity (V0) versus substrate concentration ([S]).
4.Change substrate concentration and repeat
-study relationship between reaction rate and the substrate that is being catalyzed
Km
Km=Michaelis Constant-concentration when initial velocity is half of vmax
high concentration substrate Increases
enzyme concentration becomes fixed
input in research in relationship between concentration of substrate vs. velocity
Leonor Michaelis
Maud Menton
Michaelis Menten Equation
Invertase Reaction:
sucrose + H2O glucose + fructose
V0= Vmax[S]/Km+[S]
Km»[S],
[S] at denominator becomes insignificant numerator is significant. it will be proportional vmax (s)/ km
[S]»Km
it becomes fully saturated so velocity doesn’t depend on substrate concentration. The (s) will cancel out and will V0=Vmax, you will be close or reach vmax.
Burk-plot equation
1/V0= Km/Vmax[S] + 1/vmax
x-intertecpt is negative -1/km
y intercept 1/vmax
slope= km/vmax
Intrinsic
extrinsic
intrinsic: does not depend on material (km)
Extrinsic: depends on material (vmax)
Km is an ____ property
Intrinsic property of an enzyme- the bonding affinity between the substrate and enzyme
weak bond= large km
strong bond=small km
Vmax is ___ property
extrinsic
kcat =
kcat = Vmax/[Enz] (enzyme Conc.) What does kcat mean?
kcat comes from Vmax and [Enz]
Vmax is [molar]/sec [Enz] is in molar
M-M Equation with kcat
The form of M-M equation for single substrate is
Vmax= Kcat [Etot]
kcat (turnover number): how many substrate molecules can one enzyme molecule convert per second
Km (Michaelis constant): an approximate measure of substrate’s affinity for enzyme
Microscopic meaning of Km and kcat depends on the details of the mechanism
Enzyme efficiency is limited by
diffusion:
kcat/KM
Enzyme reactions are diffusion-limited, can only work as fast as substrates gets to the active sites by molecular diffusion.
Two-Substrate Reactions
Kinetic mechanism: the order of binding of substrates and release of products
When two or more reactants are involved, enzyme kinetics allows to distinguish between different kinetic mechanisms
Sequential mechanism
Ping-Pong mechanism
Common mechanisms for enzyme-catalyzed bisubstrate reactions.
FIGURE 6–13 Common mechanisms for enzyme-catalyzed bisubstrate reactions. (a) The enzyme and both substrates come together to form a ternary complex. In ordered binding, substrate 1 must bind before substrate 2 can bind productively. In random binding, the substrates can
bind in either order. (b) An enzyme-substrate complex forms, a product leaves the complex, the altered enzyme forms a second complex with another substrate molecule, and the second product leaves, regenerating the enzyme. Substrate 1 may transfer a functional group to the enzyme (to form the covalently modified E’), which is subsequently transferred to substrate 2. This is called a Ping-Pong or double-displacement mechanism.
enzyme reaction involving a ternary complex:
-random order
-ordered
enzyme reaction in which no ternary complex is formed
Sequential Kinetic Mechanism
We cannot easily
-Random mechanisms in equilibrium will give
-Lineweaver-Burk:
-We cannot easily distinguish random from ordered
-Random mechanisms in equilibrium will give intersection point at y-axis
-Lineweaver-Burk: lines intersect
Enzyme forming ternary complexes
Ping-pong mechanism
Lineweaver-Burk: lines are parallel
Enzyme without ternary complexes
Other approaches to understanding enzyme mechanism
-Structural information such as the 3-D structure- provided by crystallography
-Genetic engineering- look at how changes in specific amino acids affect enzyme structure and activity
Enzyme Activity Inhibition: Inhibitors
Inhibitors are compounds that decrease enzyme’s activity
Irreversible inhibitors (inactivators) react with the enzyme
-One inhibitor molecule can permanently shut off one enzyme molecule
-They are often powerful toxins but also may be used as drugs
Reversible inhibitors bind to and can dissociate from the enzyme
-They are often structural analogs of substrates or products
-They are often used as drugs to slow down a specific enzyme
Reversible inhibitor can bind:
-to the free enzyme and prevent the binding of the substrate
-to the enzyme-substrate complex and prevent the reaction
Competitive Inhibition
Competes with substrate for binding
-Binds active site
-Does not affect catalysis
-No change in Vmax; apparent increase in KM
-Lineweaver-Burk: lines intersect at the y-axis
-reverssible
Uncompetitive Inhibition
-Only binds to ES complex
–Does not affect substrate binding
–Inhibits catalytic function
-Decrease in Vmax; apparent decrease in KM
-No change in KM/Vmax
-Lineweaver-Burk: lines are parallel
-reverssible
Inhibitors - Mixed Inhibition
-Binds enzyme with or without substrate
–Binds to regulatory site
–Inhibits both substrate binding and catalysis
-Decrease in Vmax; apparent change in KM
-Lineweaver-Burk: lines intersect left from the y-axis
–Noncompetitive inhibitors are mixed inhibitors such that there is no change in KM (https://quizlet.com/68124133/uncompetitive-vs-competitive-vs-non-competitive-inhibition-flash-cards/)
-reversible
Other Factors affecting enzyme activity
pH changes-
-amino acid side chains at the active site may be involved in reactions in which their ionization state is important
-pH optima varies from enzyme to enzyme but usually in range from 6-8
Temperature-
-At low temps, rate of reaction increases proportionally with increasing temp
-For many enzymes, the maximum rate is at 37ºC
-Above 50-60ºC rate falls off
Enzyme activity can be regulated
Regulation (increased/decreased) can be:
-noncovalent modification
-covalent modification (e.g. phosphorylation)
-reversible (allosteric enzymes binding with regulatory compounds (modulator or effectors); enzymes bound by regulatory proteins)
-irreversible (peptide segments of enzymes removed by proteolytic cleavage)
Regulatory Enzymes
-For a given metabolic pathway with multienzymes, the first enzyme in the sequence is regulatory – why?
-Un-necessary catalysis wastes energy and metabolites
-Generally the end product of a pathway will be inhibitory and this type of regulation is called feedback inhibition
-doesn’t have an inhibitor
-functions to regulate other enzyme’s activity
-have an active site and a regulatory site
-modulator binds to regulatory sites
Feedback Inhibition is the Classic Form of Allosteric Inhibition
-Threonine dehydratase is inhibited by isoleucine- heterotrophic allosteric inhibition
-Isoleucine only binds to the regulatory site by noncovalent binding and is reversible. When the [isoleucine] decreases, the threonine dehydratase begins to work again
Allosteric enzymes
-Regulation occurs as
-The regulatory site is
-If the modulator is the same as
-Heterotropic enzymes have
-Allosteric modulators are
-Regulation occurs as conformational changes of the modulator bind to convert different forms of the enzyme
-The regulatory site is specific for the modulator
-If the modulator is the same as the substrate then the enzyme is homotropic(substrate looks exactly like the modulator) and it is likely that the active site and regulatory site are the same
-Heterotropic enzymes have a modulator that is different than the substrate
-Allosteric modulators are not uncompetitive or mixed inhibitors
Allosteric Effectors
– Bind to Allosteric Site
Allosteric enzymes are usually larger and more complex than nonallosteric enzymes
Noncovalent Modification: Allosteric Regulators
Noncovalent Modification: Allosteric Regulators
The kinetics of allosteric regulators (Sigmoid kinetics) differ from Michaelis-Menten kinetics.
Phosphorylation Affect Structure and Catalytic Activity of Enzymes
-Addition of phosphoryl group introduces a bulky charged group into a region
-The oxygen atoms of the phosphoryl group can be hydrogen bonded to several groups, commonly the amide groups of the peptide backbone.
-The two negative charges can repel other neighboring negatively charged groups
Zymogens and activation
-they are activated by:
Zymogens (proenzymes) are inactive forms of enzymes
Zymogens are activated by removal of peptide sections, e.g. proinsulin is converted to insulin by removing a 33-amino acid peptide chain
-irreversible covalent modification
-activated when you cut peptide bonds
