Test 2 Flashcards
What are some general properties of enzymes?
- accelerate the rate of reaction of a chemical reaction by factors of 10^3 to 10^20
- Enzymes will not induce a reaction that is unfavorable due to a positive free energy change
- high specificity for the reaction substrate
- High rxn specificity
- enzyme is unchange in the reaction and can undergo rapid turnover
- subject to regulation to alter their substrate binding affinity or activity
What was the first Enzyme?
RNA
What is the largest class of enzymes?
protein -ribozymes are another class
E+S -> ES -> E + P
Label the enzyme, substrate, enzyme-substrate complex, and the product
List important points about this reaction
E= Enzyme
S= Substrat
E-S= Enzyme-substrate complext
P= Product
- the first step in this reaction (E+S-> E-S) is your slow/rate-determining step. This rate is dependent on the substrate concentration and is limited by the diffusion rate
- the second step of the reaction (ES-> E+P) is the rapid step. Once the ES complex forms the reaction takes place very rapidly thus the ES comlex does not remain long
- The second step is generally irreversible. Thus only K2 is included in the overall reaction scheme
How do enzyme change the reaction rate?
- progress of a rxn is dependent on the free energy changes as the reaction proceeds
- The enzyme lowers the activation free energy which is needed to get to the transition state, which increases the forward Rate constant
- DOES NOT change the overall Delta G of the reactoin
Transition State
an unstable arragnement of atoms with the chemical bonds in the process of being formed or broken
Activation Energy
the energy barrier of the reaction that must be overcome for the reaction to occur
Oxidoreductases
One of the 6 classes of enzymes
- catalyze oxidation-reduction (electron transfer) reactions
- typically utilize NAD-NADH and NADP-NADPH for the oxidation-reducation and electron transfer
- most frequently characterized by the gain or loss of a proton when electrons are also transfered
Oxidation
Loss of electrons
Reduction
Gain of electrons
Transferase
- one of the 6 classes of enzymes
- these enzymes transfer a chemical group from one molecule to another
- often involve coenzymes
- often involve the covalent attachment of the substrate to the enzyme
Hydrolases
these enzymes catalyze hydrolysis reactions-
-transfer functional groups to water with the cleavage of a single or double bond
What are some examples of hydrolases?
esterase, phophatase, peptidase
Lyases
enzymes catalyze (lysis) breakage of a bond,
- NO water involved
- No change in oxidation state
- Generate double bond in product
Isomerases
catalyze structural changes in a single molecule (isomerization reactions)
-always reversible but does not absolutely require stereoisomers
Ligases (synthetases)
Catalyze ligation (joining) of two substrates
- usually requires ATP as a energy source for the reaction
- ATP or its parts are not part of the product
For a reaction that needs an enzyme, what would happen to the initial velocity?
the initial velocity would depend on the amount of enzyme added as long as there is a lot of substrate for all of the enzyme concentrtions used
-Plot of V0 vs [E] should be linear as long as their is enough substrate to keep all of the enyzme molecules occupied
Initial Rate of a rxn formula?
v= k[S]
What does Vmax tell us about the enzyme properties?
the enzyme is working as fast as it can.
Vmax formula and definition
Vmax= Kcat[E]tot
- max reaction rate
- occurs when all enzyme is bound, Vmax=Kcat[E]tot
Km
Michaelis Constant
defined by the ratio of rate constants that dissociate (k1 and K-2) over what associates teh substrate (k1) but usually K2 is small compared to K-1 thus:
Km=(k2+k-1)/k1=k-1/k1
dissociation constant between the enzyme and the substrate
***concentration of S when half Etot is bound
What two parameters characterize the strength of an enzyme?
Kcat and strength of the enzyme (Km)
Kcat
Turnover number
- indicates how fast the enzyme can work under conditions in which their is saturating amount of substrate
- STRONG ENZYME =large Kcat
A small Km corresponds to what?
Strong binding (strong enzymes)
A large Km corresponds to what?
weak binding
If [S]<<<km how can the initial velocity be written>
</km>
V0= Kcat/Km[E][S]
- the rate constant for this condition is kcat/Km
- same as the second order rate constant for the reaction
Kcat/Km
enzymatic rate constant
-measures the overall efficiency of the enzyme (combination of binding strength and catalytic rate)
Diffusion Controlled limit
caused by the fact that the ES complex is formed by the diffusion S to E
-determines how small the Km can be, because the rate of the association rxn, k1, cannot be larger than the rate of diffusion of the S to E
Thus the value of Kcat/Km could not be larger than about 10^8-10^9 M-1S-1
Michaelis Menton Equation
v0=Kcat[E][S]/Km +[S]
When dealing with the Michaelis Mentin Equation, what does it mean when reactions are a Low[S]
Km>>[S]
dependence on both the enzyme and substrate thus
‘v0=(kcat/km)[E][S] fits the limiting slope
Kcat/Km can be determined
When dealing with the Michaelis Mentin equation, what does it mean when there is high [S] Values?
since all of the E are saturated at a very high [S]
V0=Kcat[E] and Kcat can be determined
Competitive Inhibition
inhibitor and substrate compete for the same binding site on the enzyme
-two versions: class and nonclassic
**in both versions, binding of inhibitor prevents the binding of the substrate
Uncompetitive Inhibitor
inhibitor binds to the ES comlex and prevents reaction
- Inhibitor decreases the Vmax/Kcat
- [ES] is lowered
- resulting in a decrease of Km/equilibrium shifts to the right
Noncompetitive Inhibition
inhibitor binds to either E or to ES complex, does not interfere with S binding but prevents reaction
- inhibitor binds to both E and Es
- Km is not changed since the biding of S is not inhibited
- Vmax and kcat is decreased
Competitive Inhibition
the inhibitor and substrate compete for the same binding site on the enzyme.
-two versions classic and nonclassic
***both versions the binding of the inhibitor prevents binding of substrate
**inhibitor lowers concentration of available E, resulting in an increase of Km/equilibrium shifting to the left
If there is a high concentration of [S] in competitive inhibition what results?
high [S] overcomes the effect of the inhibitor, thus Vmax is not chagned
What type of inhibition is this?
Simple End product inhibition
- the final product inhibits the first enzyme in the pathway
- Frequently called FEEDBACK INHIBITION
Sequential End-product inhibition
- E and G individually control their own synthesis so they will not interfere with synthesis of each other
- The arrangement of this pathway allows the continuing synthesis of either E or G, if either of those substances are specifically needed.
What type of inhibition is this?
Concerted End-product inhibition
-synthesis of the common precursor D for the E and G pathways is slowed down whenever either pathway is inhibited
DIfferential Inhibition of multiple Enzymes
Substrate Activation
- accumulation of substrate A induces activation of the enzyme to increase synthesis of B and activating the following pathway
- Typically Allosteric activation involving a multi subunit enzyme exhibitying cooperative substrate binding and alteration of enzyme structure to increase substrate binding interaction with the enzyme
Enzyme structure and activity can be influenced by what?
interaction with small molecules called effectors
What can be an effector?
enzyme’s substrate
enzymes products
often other effector molecules that do not resember substrate
Allosteric Enzymes
multi-subunit enzymes
-Do not exhibit Michaelis Mentin Kinetics instead exhibits a sigmoid kinets (s shaped)
Multi-Subunit Enzyme
binding of the first (and subsequent) effector to a subunit:
induces structural changes in the subunit and in other subunits
this alters the binding strength of the substrate (a measure of this is an altered Km in the subunits)
Allostery
the capability of one ligand to bind and affect the binding of the second ligand at a distant site
Positive Coopertivity
-Draw a relative graph of what would happen
binding of the first ligand increases the affinity of subsequent ligands
Negative cooperativity
-Draw a relative graph of what would happen
binding of the first ligand decreases teh affinity of subsequent ligand
Allosteric Activator
ligand that increases activity of the enzyme
Allosteric Inhibitor
is a ligand that decreases the activity of the enzyme
If the effector is in an inhibitor what will happen?
it will increase the tendency to the T state(low affinity for substrate)
If the effector is an activatory what will happen?
it will increase the tendency to the R state (high affinity for substrate)
ATC
aspartate transcarbamoylase
- classic example of allosteric regulation of an enzyme in a key metabolic pathway
- catalyzes the synthesis of the pyrimidine ring needed for all the pyrimidine nucleosides in the cell.
- ATC is 12 subunits – 6 regulatory, 6 catalytic
-
when dealing with ATC, if there is excess CTP what will happen?
ATC will be inhibited
When dealing with ATC, if there is excess ATP what will happen?
ATC will be activated to make CTP to catch up to [ATP]
Enzyme Regulation by Covalent Modifications
- most common types are methylation, phosphorylation, and acetylation
- regulate enzymatic activity by changing the R to T equilibirum
-
Pyruvate Dehydrogenase
- involved in glycolysis and is subject to many control mechs
- Pyruvate Dehydrogenase is deactivated when phosphate is attached and the enzyme specifically carries out the phosphate addition
Pyruvate Dehydrogenase Phosphatase
specifically removes the phosphate from pyruvate dehydrogenase
How can an enzyme increase the reaction rate?
by lowering free energy of transition state
- binding the substrates A and B (this raises the free energy, so it makes the activation free energy smaller)
- bind A and B and also bind to the transition state to reduce its free energy
Limit to binding strength (Km) values for enzyme
enzymes need to have low Km values to have good affinity to their substances and good specificity to prevent inappropriate products
Why shouldn’t enzymes bind to their substrates to tightly?
- tight binding could prevent reaction, substrates bound in very tight binding pockets will be incompatible with reaction
- strong binding could result in a E-S intermediate which would have an even harder time reaching the transition state than the uncatalyzed reaction
- Very tight binding pockets could prevent product release
Significance of Induced Fit Binding Mechanism
Enzymes are flexible and the ability to collapse on a suitable substrate offers several advantages
- the substrate changes the enzyme structure and this can be connect with the enzyme activity (ex: the enzyme is not active unless the correct substrate is present)
- the enzyme structure is fairly open and accessible to the substrate, until it binds; this allows access but prevents water and other undersirable molecules from the active site
- after reaction, the enzyme can reversibly change its structure and release product
Organization of enzyme active sites
active sites for enzyme catalysis will generally be in interior locations
–Many reactions will need to exclude water, or carefully control it, at the reaction site
-Want to have a large area of contact between substrates and enzyme for substrate specificity
Many amino acids lining an active site will be nonpolar
- this will help exclude water from the active site
- nonpolar enviroment will hlep raise electrostatic effects in active site
***However, there will be key polar and charged amino acids at the active site that will be involved in binding the substrates and in the reaction mechanism;
Nucleophile
a functional group rich in, and capable of, donating electrons
Electrophiles
a functional group that seeks electrons
What are the amino acids that act as bases
His, Asp, Glu
What are the amino acids that act as acids
His, Lys
How does base catalysis happen?
Directly
-extraction of proton from the substrate to activate the reaction indirectly through water-extraction of proton from water generates OH
When does acid catalysis happens
happens usually through direct protonation (addition of H+) to the substrate to activate the reaction
Covalent Catalysis
- an intermediate is formed by the covalent joining of the substrate and the enzyme
- This intermediate goes on further
Effect of pH changes on reaction rates
pH of the solvent can significantly impact the activity of the enzyme
- as the pH increases or decreases the ionization states of the amino acid side groups change
- If critical side groups are in the active site, changes in ionization will alter the strength of substrate binding or the catalytic acitivty
-
Transition state stabilization
very tight binding between the reactant and the enzyme in the transition state which helps lower the transition state energy
Transition State Analog
a chemical compound that resembles a transition state arrangement
