Biochemistry Exam 2 Flashcards
exergonic reaction
releases energy as a part of the reaction
endergonic reaction
needs energy for the reaction to take place
Enzymes are proteins that catalyze chemical runs by
Binding Site Ligand Specificity Temperature sensitivity pH Sensitivity
Binding Site
active sires are binding sites that contain residues with high affinity for substrate and are the site of catalysis
allosteric sites are ligand binding sites that influence enzyme conformation and thus catalytic activity
Ligand
ligand is the reactant and the substrate, or just the molecule that binds to active site and undergoes chemical change
allosteric regulator is a molecule that binds an enzyme outside the active site and influences catalytic activity
Specificity
Specificity varies with enzyme, some enzymes have a single active site that is highly specific for one form of ligand, while other enzyme active sites can accommodate multiple substrates
Examples of specificity
Hexokinase- enzyme that has 4 isozymes
Hexokinase I/A has a wide tissue expression, and catalyzes hydrolysis of ATP followed by phosphorylation of a hexose in the reaction
Hexokinase IV/D or glucokinase - selectively expressed in liver, pancreas, smal intestine, and has a lower affinity for glucose
it only catalyzes rxn with glucose
Isosyme
catalyzing the same chemical reactions but all have different amino acid sequences, tissue expression patterns, affinity for substrate
Temperature Sensitivity
each enzyme has an optimal temp. humans is at 35-40 degrees C. Higher temp results in faster molecule motion and catalysis where as cooler temp results in slower motion and catalysis
too much heat will denature a protein
pH Sensitivity
active sites contain amino acid residues with distinct ionization stats
altering the hydrogen ion concentration in solution will alter the ionization and therefore denature the protein
Apoenzyme
the protein portion of the enzyme
Prosthetic Group
chemical component, either a metal ion or molecule is called the prosthetic group.
it is usually covalently bonded to the protein
How enzymes work
enzymes catalyze a reaction by increasing the rate at which a reactant is converted into product
the key to catalysis is the formation of ES, enzyme plus substrate
Proof of ES existing before P
electron microscopy and x-ray crystallography
physical properties of an enzyme change upon binding substrate
high specificity for substrate
ES can be isolate in pure form
when enzyme is saturated with substrate, the rate of product formation is constant
Enzyme Thermodynamics
catalyze the conversion of energy forms
there must be a energy source to do the work of conversion
-dG is spontaneous, +dG is non-spontaneus
Ways to drive run are by energy coupling and equilibrium influences
Energy Coupling
combine a chemical reaction with excess free energy, or -dG with a +dG rxn
dG
the magnitude of G depends on how far from equilibrium you system is initially
Keq
equilibrium constant reflecting molar concetration of product/reactant at equilibrium
if Keq is large then the reaction favours the products and is more likely to go to completion
if Keq is small then the reaction favours the reactants and is less likely to go to completion
S–>P
goes thru a transition state, in which the molecule is no longer S or P
the energy create the transition molecule is not calculated in the final dG b/c this energy is release upon making product
we get a value of energy that represents the amount needed to activate the rxn known as energy of activation
E + S going to ES
substrate binding to the active site lowers the activation
it is down by multiple weak non-covalent bonds that release energy upon substrate:active site binding, satisfying the energy debt by enzyme
Structure of the Active Site
binds substrate, but not in a perfect complementary fit
with non-covalent bonds, conformation of both substrate and enzyme change forming induced fit
as substrate is encountered, binding energy is low and reaches a maximal binding energy to satisfy activation energy debt
enzyme thus most complementary to the transition state
Active Structure dictates function
non-covalent binding energies are driving force of catalysis
microenvironment of active site is non-polar, unless water is used as a substrate
-in a non-polar envirnment, polar residue have special catalytic or binding properties
active site residues are oriented due to 3D folding; this gives more range of motion in the active site compared to steric hinderance seen in primary structure
1st order reaction
when the rate of reaction is directly related to concentration of substrate
Zero order reaction
when rate is independent of substrate
usually when substrate is much greater than enzyme
Second order reaction
when rate is dependant on 2 substrates
Steady State Assumptions
- consider only the forward reaction
- measure rate from initial introduction of enzyme
- enzyme concentration is constant
- substrate is typically greater than enzyme concentration
- concentration of ES stays the same
- Vmax can’t be obtained b/c of diffusion, temp, pressure
E + S ES
is a fast reversible reaction
rate of formation is k1
rate of dissociation is k-1
ES E +P
is a slower reversible reaction
rate of formation is k2
rate of dissociation is k-2
What is the overall reaction rate proportional to
The concentration of ES, the product formation is the rate limiting step due to slower reaction of ES to P
due to the steady state assumption we assume negligible product reversion to substrate, so k-2 approaches 0
How do we know ES proceeds to P
initial reaction rate is dependant on rate productiomn formation of ES
ES can be formed and broken down, and in steady state k values for ES are equal
k1 [E][S] = (k-1+k2)[ES]
M and M developed a rate constant to represent the proportion of ES breakdown/formation, km
we cant measure [ES], but in vitro we have a known [E]t that is equal to the amount of [E] and [ES] together
Michaelis and Menton equation vs. Lineweaver Burk
from the M and M algebraic equation gives a hyperbolic curve. To predict km and Vmax using MM equation you need several points to make an accurate line
Lineweaver Burk is a double reciprocal plot of MM equation that creates the equation y=mx+b, and for this graph is requires fewer data points and you also know the x and y intercepts
Using km and kcat
km reflects the affinity for an enzyme for substrate ONLY when k2 is much smaller than k-1
if the dissociation of enzyme and substrate together is high, then affinity of enzyme for substrate is low
if k2 is greater than k-1, then km is not a measure of affinity
As enzyme kinetics grow in substrate and transistion states, the number of rate constants increase
Not all enzymes have the same RLS and can be dependant on each other, kcat measure the overall rate of catalysis
kcat/km is a measure or catalytic effeciency
by determining the ratio of kcat/km we know the rate of catalysis and affinity, most use is comparing an enzyme preference for substrate
The most efficient enzymes have limitations to Vmax due to diffusion in water
Allosteric Enzymes
do not obey MM kinetics
have multiple binding sites on a single enzyme or holoenzyme that influences the velocity of enzyme activity in the presence of substrate
The two type of allosteric enzymes are homotropic and heterotropic
Homotropic Enzyme/Allosterism
Binding of one substrate alters the conformation of the enzyme such that the second substrate binds more cooperatively
Converting from T to R state with each bound substrate increases the velocity, creating a sigmoid curve
Cooperativity allows for more regulation
Heterotropic Enzyme/Allosterism
non-substrate molecules binding outside the active site to influence velocity
they can be positive or negative
in stimulation the R state is favoured, Vmax/2 is achieved with less substrate
in inhibition the T state is favoured, Vmax/2 is achieved with more substrate
Inhibition
enzymes can be inhitbited by specific molecules, altering catalytic rates measured by classic MM kinetics
molecules can be reversible when they are joined by non-covalent interactions, the types of inhibitors have a rapid dissociation from the [EI] complex
molecules can be irreversible when they are joined with covalent or strong non-covalent bonds, key is the inhibitor dissociates at a very slow rate
eg. Suicide inhibitor such as penecillin form covalent bonds at the catalytic site and prevent further use of enzyme
Inhibitors such as asparin make a covalent modificattion to cyclooxygenase
Competitive Inhibition
A competitor [I] competes with substrate for the active site
- inhibitor resembles the substrate
- no product is formed
- does not change the Vmax b/c sufficiently high [S] can overcome inhibition
- creates apparent increase km making rxn slower b/c it appears the affinity for substrate is reduced when reality says enzyme is bound by inhibitor
on the LIneweaver Burk graph the Vmax does not change but the km increases and moves towards the y axis (x increases)
Naturally Occuring competitive examples
Digitalis from foxglove bind to NaK ATPase
Tetradotoxin from puffer fish binds Na channels
Cytochalasin B from fungus binds glucose transporter
Atropine from nightshade binds acetycholine receptor
Synthetic Competitive Inhibitors
Sulfanilimide is an antibacterial that inhibits folate synthesis
Neostigmine inhibits acetylcholinesterase, prolongs neuromuscular transmission in diseases like myasthenia gravis
Indinavir inhibits HIV protease II to treat HIV infection
Uncompetitive Inhibition
Inhibitor binds the ES complex, at site distinct from the active site
- inhibitor only binds ES complex, so if [E] > [I] some enzyme activity will occur
- Vmax decreases b/c functional [E] decreases
- Apparent km decreases b/c ES is functionally inactive. As less active enzyme is available, less substrate is required to load enzyme and thus apparent km decreases
In the Lineweaver Burk the km decreases (moves from y axis, x decreases) and the Vmax decreases (moves up y axis)
Uncompetitive Inhibition Example
Glyceraldehyde 3-phosphotase Dehydrogenase (GAPDH) is one of 10 enzymes in the process of glycolysis (6th step)
GAPDH binds 3 substrates in the precise following order ebfore making product, glycerate 1,3 biphosphate
-NAD+
-Glyceraldehyde 3-phosphate
-PO4-
AsO4- is an uncompetitive inhibitor b/c it has similar shape as PO4- and b/c PO4- is the last in the order above the ASo4- binds the ES complex
Non-Competitive Inihibtion
Inhibitor and substrate bind simultaneously at distinct binding sites, thus inhibitor binds either the E or ES complex
-Vmax decreases b/c functional enzyme is depleted from solution
km is unchanged b/c active site is not altered
-common theme is feedback inhibition
On the Lineweaver Burk the km does not change bu the Vmax is decreasing (moves up the y axis)
Non-competitive example
Heavy metal poisoning causes wide spread denaturation of proteins (Lead, Mercury and sliver). React with sulfhydryl groups on cysteines to cause misfiling of tertiary structures of proteins
Naturally occuring- Caffeic acid found in tomatoes inhibit lypooxygenase
Caffeine found in tea and coffee inhibits cAMP phosphodiesterase Synthetic
Synthetic - trichostatin A inhibits histone acetyltransferase and is anti-cancer
Mycophenolic acid inhibits inosine monophosphate transferase and used to treat flavivirus infections like west nile virus and dengue virus
Suicide Inhibition
Compounds that bind to the active side of an enzyme and undergo chemical steps of the reaction, but form a covalent or stable non- covalent bond with the enzyme
-compounds would be substrate analogs, and thus you would not anticipate competitive inhibition graphs
-since suicide inhibitors inactivate the enzyme, Vmax decreases
-suicide inhibitors do not affect the km
Therefore, suicide inhibitors would have a graph similar to non-competitive b/c inhibition cannot be relieved at high substrate concentrations
Suicide Inhibtion of Aspirin
Aspirin as a substrate analogs for cyclooxygenase-1 (best inhibition) and cyclooxygenase-2 (induced specifically by cytokines and other immune stimulation).
COX enzymes normally bind archaadonic acid to make prostaglandins (PGG) which are pro-inflammatory precursors
Aspirin acetylates a serine residue in the upper channel of the active site preventing arachodonic acid binding
Suicide Inhibition of Monoamine Oxidase
MAO is important for breaking down dopamine and seratonin. In disorders such as Parkinson’s and depression, which less neurotransmitter is made, inhibiting MAO is therapeutic leaving the dopamine and seratonin in the system longer
N,N dimethylpropargylamine makes a covalent bond with the flavin prosthetic group in MAO
Catalytic Strategies
Enzyme catalyzed reactions begin with substrate binding
Binding Energy not only ensure optimal structural associations, but also facilitates catalysis
Induced fit dictates that the ES complex is oriented such that the nucleophiles are able to attack electrophiles, and the electrophile is in approximation with the nuc.
4 Types of catalytic Strategies
Covalent catalyis - the active site contains a powerful nuc that leads to a covalently bound transition intermediate
General Acid-base catalysis - a molecule in the active site other than water is the proton donor/acceptor.
Metal Ion catalysis - a metal ion may facilitate the formation of nuc, or the metal ion itself may be the elec. or nuc. A metal ion may also participate in increasing the binding energy by orienting the substrate for catalysis.
Catalysis by approximation - ensures that 2 distinct substrates are localized together along 2 active sites of an enzyme
The Protease
Protease are enzymes involved in digestion, but also in AA scavenging at protein turnover
All proteases us the process of hydrolysis
-the covalent peptide bond and the covalent H-OH bond is broken. The unpaired electrons resulting from the broken bonds are covalently joined, such that the H bonds with the amino group and the OH bonds with the carbonyl carbon
Proteins are more stable than other molecules containing carbonyl groups b/c the bond resonance about the peptide bond stabilize the bond as well as decrease the reactivity of the carbonyl carbon
Chymotrypsin as a Serine Protease and the 3 components
Chymotrypsin uses 2 catalytic strategies: acid/base and covalent catalysis 3 components of the active site: -hydrophobic pocket -oxyanion hole -catalytic triad
Hydrophobic pocket
to attract Trp, Tyr, Phe or Met.
the hydrophobic pocket aligns the protein such that the peptide bond between the hydrophobic residue and the next residue is oriented next to the catalytic site of serene.
