topic 05 enzyme catalysis Flashcards
define substrate
molecule on which an enzyme acts
the names of enzymes often end with?
-ase
-in
-zyme
what is special about enzymes?
specificity: will recognize one/few molecules out of thousands
accurate/fidelity: almost never makes mistakes. resulting in almost a 100% yield
rapidity: accelerates reactions greatly
ability to work under mild conditions: such as low temperatures/pressures, neutral pH, etc - however, they do work best under specific conditions
regenerate: they are regenerated during the reaction
describe the active site of an enzyme
cleft or crevice between domains
define transition state
the state, in a chemical reaction, in which it has the highest energy. once this state has been achieved, the reaction is irreversible
it is the intermediate form between reactants and products
what must reactants over come to form products? (what’s the symbol)
energy barrier: ∆G≠
what are the mechanisms enzymes employ to lower the activation energy? which method is the most common?
A) enzyme binds to 2 substrate molecules and orient them in a way to encourage a reaction
B) binding of substrate to enzyme rearranges the electrons in the substrate, creating partial negative and positive charges that encourage a reaction
most common method
C) enzyme strains the bound substrate molecule, forcing it toward a transition state to encourage a reaction
define transition state analogues. give example
enzyme inhibitors that resemble transition states but are not reactive
many drugs and antibiotics are inhibitors
describe enzyme cofactors. what are some enzyme cofactors (what form do they come in/what do they consist of)?
a required partner that some enzymes (apoenzymes) require to function. they may be essential but they also increase the rate of reaction
enzyme cofactors could be proteins, metal ions, or co-enzymes (organic/metallo-organic molecules derived from vitamins). most consist of protein or RNA or a complex of RNA and protein
define apoenzymes
an enzymatically inactive protein part of an enzyme that requires a cofactor for its activity
what do enzymes not affect?
equilibrium constant K: the ratio of substrate to products
different in free energy between reactants and products (∆G), this is because ∆G is related to K
what has the highest affinity for a reaction’s transition state?
enzymes
…
NOT substrates or products
what are the strategies used by enzymes to attain transition states?
bringing substrates together (in multi-substrate reaction)
orientating substrates in a favourable geometry
supplying proton acceptors/donors & electron donors/acceptors
excluding water (in some cases)
stressing substrate physically or electronically
selective binding of the transition state
what may happen to enzymes under extreme conditions such as pH and temperatures?
extreme pH
enzymes may be protonated/deprotonated or may denature
extreme temperature
enzymes may become unstable due to increasing reaction rate
what is the catalytic triad of serine protease made up of?
Serine, Aspartate, and Histidine
what could found in the active site of a serine protease?
critical serine residue
point out the differences and similarities between the two phases the serine protease undergoes while explaining the process
phase one
1. hydrolysis: serine performs nucleophilic attack on the carbonyl-carbon (carbon of carbonyl :3) of the peptide bond, resulting in a scissile bond.
2. the nucleophilic attack is facilitated by Histidine 57 which acts as a general base to remove proton from serine (serine normally can’t release a proton). Histidine is a strong base because of the hydrogen bond with aspartate, a member of the triad. This results in a tetrahedral intermediate with a substrate that is covalently bonded to enzyme through the serine
3. tetrahedral intermediate has high energy and is unstable, therefore, it will decompose when Histidine 57 acts as an acid and gives back the proton to the nitrogen of the scissile peptide bond. This bond then dissociates, resulting in an acyl-enzyme intermediate which has 1/2 of the cleaved substrate.
4. remaining steps break the bond, release the remainder of the substrate, and regenerate the enzyme
phase two
1. hydrolysis: water attacks carbonyl-carbon of acyl-enzyme intermediate
2. the water attack is facilitated by Histidine 57 which acts a general base to remove proton from water. resulting deprotonated oxygen attacks the carbonyl-carbon. resulting in a tetrahedral intermediate (similar to phase one’s)
3. Histidine acts as acid and donates proton to serine’s active site to regenerate the original hydroxyl form and to release remainder of product
in the process of the serine protease, what is the transition state?
tetrahedral intermediate
in the process of the serine protease, what does the decomposition of the tetrahedral intermediate result in? (for both phases)
phase 1: formation of the acyl-enzyme intermediate and release of carboxyl-terminal half of the substrate
phase 2: release of amino terminal half of substrate and regeneration of original enzyme
in regards to transition state, what is a major feature of enzymes?
enzymes can stabilize transition states
describe the oxyanion and its process in regards to the tetrahedral intermediate
the tetrahedral intermediate contains an unstable oxyanion on the carbon
for the intermediate to be formed, the oxyanion on the carbon must be stabilized by an enzyme
when the tetrahedral intermediate forms, the oxyanion enters the oxyanion hole and then forms hydrogen bonds between the Histidine and Aspartate side chains which shorten - stabilizing it
describe the specificity pocket
the specificity pocket allows for cleaving after different residues. it binds to residues and substrate that the enzymes specifically recognizes. this binding positions the peptide (scissile) bond optimally for cleaving by the active site residue.
describe the specificity pocket for chymotrypsin and trypsin and elastase
chymotrypsin: must recognize bulky aromatic residues. serine at the base of pocket and glycine lining the pocket to make room for bulky side chains
trypsin: must recognize basic amino acid side chains. aspartate residue at base, allowing ionic interactions with the substrate
elastase: must recognize small aliphatic side chains. more bulky side chains linking the pocket
what are serine proteases usually synthesized as? what is the purpose of this? give another example of this?
inactive precursors of an enzyme aka zymogens aka proenzymes
this allows enzyme activity to be controlled to prevent damage that may arise if left alone
another example of zymogens: in blood coagulation, all enzymes involved are circulating in the blood as zymogens. they only get activated when a wound occurs, at the site of the wound. otherwise, there would be uncontrolled coagulation through out the blood stream
describe the activation process of trypsin and chymotrypsin
activated after secretion after being cleaved in 1 or 2 places by specific proteases
the trypsin is cleaved by enteropeptidase. the trypsin goes on to activate the chymotrypsinogen (zymogen form of chymotrypsin). chymotrypsin can also act on itself to make a few more clips but all of the cleaved forms are active – even the clipped structure retained through disulfide bonds
at a fixed enzyme concentration, what is the relationship between the rate of the reaction and substrate concentration?
the rate of reaction will increase as the substrate concentration increases until a limit is approached.
as the enzyme becomes further saturated with substrate, further increases in substrate concentration does not increase the rate of reaction
what is the michaelis-menten equation? when does this equation apply?
Vo = Vmax [S] / (Km + [S])
Vo - initial velocity
Vmax - maximal velocity (velocity at infinite substrate concentration at a given concentration of the enzyme)
S - substrate concentration
Km - michaelis constant. it equals the substrate concentration required to give a rate that’s half of Vmax
this equation only applies when the concentration of the initial complex is constant
in the michaelis-menten equation, what is Vmax dependent on?
the Vmax of a reaction directly depends on the enzyme concentration (doubling the amount of enzyme will double the rate of product formation)
describe Km and its relationships and relevance. why is it not an equilibrium constant?
Km is not dependent on the enzyme concentration. however, it is inversely related to the affinity of the enzyme for the substrate. (lower Km = higher affinity)
when the substrate concentration = Km, the rate is 1/2 the max rate (Vmax)
Km is not an equilibrium constant because although substrate binding is reversible, some of the bonded substrate turns over into the product
how would one use the michaelis-menten equation?
plot equation using “double-reciprocal” plot or “lineweaver-burk” plot which
gives a straight line
double-reciprocal equation
1/V = (Km/Vmax)(1/[S]) + (1/Vmax)
this allows determination of Km & Vmax from velocity vs [Substrate] data on a graph y=mx+b
what are the two classes of inhibitors?
irreversible: covalent bond with enzyme
reversible: non-covalent bond
what type of inhibitor is penicillin?
irreversible
inhibits enzyme that contributes to cell wall synthesis - therefore, inhibiting organism’s growth
describe reversible inhibitors
aka competitive inhibitors
binds directly in active site and blocks substrate binding
describe the kinetic effect
increasing Km without affecting Vmax
this type of inhibition can be overcame with high substrate concentration
how can the strong activity of enzymes be controlled?
control of enzyme abundance (through expression of its gene)
synthesis of enzymes as inactive precursors (zymogens) that must be activated
reversible modifications such as phosphorylation
allosteric regulation
how do allosteric enzymes bind?
reversible binding of ligands at locations in the enzyme remote from the active site, that leads to alterations in enzyme activity.
similar to how protons bind to hemoglobin to promote T state
describe allosteric regulation
process in which proteins transmit effects of binding at one site to another, often distal, functional site, allowing for regulation of activity - cooperativity!!
binding of regulatory molecule changes conformation of enzyme and affects its activity
usually, one enzyme in the pathway is regulated - the activity of this key enzyme would be repressed by the product of the pathway (negative feedback) & accelerated by precursors
allosteric regulators bind to enzyme or its regulatory subunits to promote T/R states
important feature of metabolic pathways
what is required for cooperativity?
enzyme must have more than 1 subunit & more than 1 active site
what example is given for allosteric regulation? describe it
Aspartate Transcarbamoylase (ATCase)
catalyzes in early steps of biosynthesis of nucleotide CTP
has catalytic subunits containing active sites and regulatory subunits which bind regulatory molecules (ATP & CTP)
it’s R state is more active
Michaelis-Menten doesn’t apply but Km & Vmax can still be measured
when graphed, what does the chart of allosteric regulation yield?
its chart yields an S-curve (similar to hemoglobin) because as Aspartate concentration increases, more of the enzyme is shifted from the less active T-state to the more active R-state. when CTP binds, it slows down the enzyme, shifting curve to right. when ATP binds, speeds up enzyme.
in allosteric regulation, what happens as the Aspartate concentration increases?
as Aspartate concentration increases, more of the enzyme is shifted from the less active T-state to the more active R-state. eventually, the enzyme becomes saturated with substrate. the cooperative behaviour allows rate of reaction to be controlled over wider ranges of substrate concentrations. if it wasn’t a cooperative enzyme, activity levels would shoot up to the max level even at low Aspartate concentrations
why do metabolic pathways need to be controlled?
so right amount of synthesis occurs at the cell’s given conditions
