Enzymes Flashcards
Structure of Enzymes
active site = small amount of amino acids with catalytic side chains that come together in tertiary structure and form a cleft. binds the substrate noncovalently bind the substrate STRONGLY
*nonpolar and excludes water
other amino acids form a scaffold to keep active site in the correct conformation
Induced fit model
enzyme is inactive until the substrate binds to it and induces a conformational change which activates the enzyme
lock and key model
enzymes have a rigid, predefined active site that perfectly matches up with the substrate and forms the ES compplex
this is wrong! doesn’t explain why some substrates perfectly fit into enzymes and don’t react
How do enzymes accelerate the rate of reactions?
Enzymes bind to the substrate during the transition state and create a new reaction pathway with a lower activation energy for the reaction to proceed. Lowering the activation energy allows more molecules to reach the transition state and proceed.
enyzmes do not change the free energy of the reaction or the reaction equilibrium (∆G)
Things that effect enzyme activity and rate
temperature- increases rate/activity because more collision between E and S (if temp too high will denature it)
pH- can alter the ionization state of the substrate or enzyme catalytic groups and if too high, it will denature the protein
Catalytic strategies
**proximity **(inc effective concentration and correct orientation of substrate A w/ B)
**transition state stabilization **(oxyanion hole)
**covalent catalysis or nucleophilic catalysis **(serine binds to acyl group)
**general acid-base catalysis **(creates potent nucleophile serine)
metal ion catalysis
Chymotrypsin protease action in steps
step 1
CATALYTIC TRIAD: aspartate-histidine-serine
aspartate COO- binds to H-N on one side of histidine changing its pKa
histidine N (general base) on other side pulls the H off of Serine’s hydroxyl group creating an alkoxide ion (acid-base catalysis)
chymotrypsin protease action in steps
step 2
potent nucleophile seriene attacks the carbonyl bond of the amide forming a tetrahedral intermediate **which is stabilized in the oxyanion hole by hydrogen bonds on the enzyme (covalent catalysis)
*this tight binding is only possible in the transition state
chymotrypsin action in steps
step 3
HisH+ donates a proton (general acid) to the amine portion which falls off and the acyl enzyme is created between the oxygen of serience and the carbonyl portion of the peptide
chymotrypsin action in steps
step 4
water hydrolizes the bond and the carboxylic acid leaves
How do endergonic reactions proceed?
∆G > 0 so not spontaneous
(products have more energy than reactants)
so you can drive it forward by using ATP or coupling it to an exergonic reaction
example) couple ATp hydrolysis (∆G = -30.5 kJ/mol) with glucose phosphorylation (∆G = 13.8 kJ/mol)
energies are additive: ∆G = -16.7 kJ/mol is still spontaneous
Enzyme Kinetics: v versus E and v versus S
enzymatic rate increases linearly with enzyme concentraion
enzyme increases asymptotivally with increasing substrate concentration (reach a v max because saturated ES complex)
Enzyme kinetics: Michaelis Menten Equation
v = Vmax [S] / Km + [S]
where:
v = initial velocity at a given substrate concentration [S]
Km is a contant (how tightly S binds to E, the tighter the binding, the smaller the Km and the less S needs to be used)
kcat x [E] = Vmax (higher the kcat, the faster the reaction)
S where Vmax/2 = Km (x intercept on hyperbolic plot)
Enzyme Kinetics: Lineweaver-Burk plot
1/v = Km/Vmax[S] + 1/Vmax
in y = mx + b form
x intercept: -1/Km
y intercept: 1/Vmax
What is Km?
units: M, mM, uM
it is a measure of the affinity of the substrate for the enzyme (i.ehow tightly do they bind…the more interactions between E and S there are, the lower the Km)
each enzyme has their own Km
constant but does change with pH and temperature
Vmax and kcat
Vmax: everything is ES, maximum velocity with which the enzyme can catalyze the reaction
kcat: Vmax/[E]. Turnover number of an enzyme reflecting the number of moles of substrates converted to products per second per mol of enzyme
kcat is constant and only changes with pH and temperature
(Brief) How enzymes can be regulated
extracellular signal –> TF –> protein
(mRNA can be degraded or protein can be degraded)
Enzyme can combine with a regulatory protein or be turned on and off with PO42- (covalent modification of AA)
Enzyme can bind to a ligand acting as an allosteric effector
Enzyme can be sequestered in subcellular organelle
Enzyme phosphorylation and dephosphorylation
phosphorylated via protein kinase, makes very negative and changes catalytic activity
dephosphorylated via protein phosphatase
reversible reactions
Enzymes regulated by specific proteolysis
irreversible way to activate enzyme
ex) digestive system, blood clotting, insulin, collagen, apoptosis via caspases
ex) pancreatic zymogens (pepsinogen cleaved to form pepsin* which is now active)
Enzyme Inhibition
irreversible
molecules covalently bind to the enzyme to inhibit activity
these inhibitor molecules look like the natural substrate
ex) penicillin bonds with transpeptidase preventing the synthesis of bacterial cell wall
Enzyme Inhibition
reversible inhibition
1) competitive: binds to same active site as substrate. looks like the substrate but doesnt react
2) noncompetitive: binds to a separate sitre from the active site and causes a conformational change in the enzyme at the active site
Competitive vs Noncompetitive inhibitors
kinetics that distinguish them
competitive inhibitor: Km increases (bc doesnt let substrate bind to the enzyme so Km increases), Vmax doesnt change because a high [S] overcomes inhibition
noncompetitive inhibitor: Km doesn’t change but Vmax decreases (bc you changed the catalytic activity of the enzyme)
High [S] doesnt overcome inhibition
Competitive vs Noncompetitive Inhibition
How do you distinguish them on a LB graph?
competitive inhibitor: enzyme in the presence of inhibitor will show the same intercept on the y axis as the regular enzyme but will have a smaller x intercept because it is the reciprocal
noncompetitive inhibitor: enzyme in the presence of inhibitor will have a different y intercept than the regular enzyme (it will be higher because it is reciprocal) and they will converge at the same x intercept
How can you make a POTENT competitive inhibitor? i.e. an inhibitor that is really good at its job
TRANSITION STATE ANALOG
why? here it is stable
recall- enzymes bind more tightly to the transition state substrate than the regular substate.
as a result. the enzyme will bind really tight to the transition state analog competitive inhibitor
Feedback Regulation (involving enzymes and products)
metabolic pathways are regulated
for example) enzyme 1 in the pathway may be allosteric and two much product E downstream will bind to enzyme 1 and inhibit it (stopping the entire pathway)
or, enzyme T further down in the may activate enzyme 1 and effect it in this manner
Allosteric enzymes
properties
these enzymes have an active site and an allosteric site
they are oligomeric (i.e. at least a dimer or some sort like Hemoglobin)
active site binds S and the allosteric site binds the effector or allosteric molecules that regulate enzyme activity (allosteric molecules don’t look like substrate)
Allosteric Enzymes
How can they be kinetically distinguished?
does not show a hyperbolic v versus [S] curve
instead show a sigmoidal curve
why? cooperative substrate binding meaning that the binding of the substrate at one point on the molecule alters the binding of the substrate at another part of the molecule
ex) hemoglobin
Allosteric Enzymes
mechanism of regulation
they exist in 2 confirmations: active R-state (relaxed) and inactive T-state (tense)
R has a high affinity for S (T is opposite)
why? substrate binding stabilizes the molecule but there are many active sites. It is difficult for the first S to bind to the T state. Once the 1st S binds, the molecule opens up and all substrates can bind to an active site
Allosteric regulators/effectors modulate the T to R equilibrium
allosteric activators- stabilizes the R-state which increases S binding (which can increase activity)
allosteric inhibitors- stabilizes the inactive T state which decreases S binding (which can decrease activity)
Allosteric Inhibitors
K type and V type
K type: increases the Km (makes substrate binding weaker) but does not affect Vmax
at low [S], the activity of enzyme is lower than normal and at high [S], activity of the enzyme is normal
V type: decreases the Vmax but will not affect the Km. The affinity of S for E is not affected.
At all [S], the activity of E is lower
Allosteric activators
K type and V type
K type: decreases Km (increasing strength of S to E bond), will not effect Vmax
at low [S], activity of E is higher and at high [S], activity of E is normal (saturated ES complex)
V type: increases V max and doesn’t effect Km
at all [S], the activity of E is higher
What type of allosteric inhibition is this?
K type
What type of allosteric inhibition is this?
V type
What type of allosteric activation is this?
K type
What type of allosteric activation is this?
V type
Enzymatic cofactors
Nomenclature
apoenzyme + cofactor (or coenzyme) = holoenzyme
holoenzyme- active enyme with non protein component
without active component? aponzyme (a means without)
cofactor- metal ion i.e. Zn or Fe
coenzyme- small organic molecule that can be a) cosubstrate if transient (NAD+) or prosthetic group if permanent (heme)
