MCAT - ENZYMES
Terms in this set (34)
How do enzymes affect the rate of a reaction?
Enzymes just increase the RATE of a FAVORABLE reaction.
Does not make the reaction more favorable energetically. Does not change the equilibrium constant.
Enzymes do NOT alter the the ΔG°' (standard free energy change) or Keq. They accelerate how fast the reaction reaches the equilibrium but don't change Keq or the free energy; that's a function of the bond energies and the concentrations. Equilibrium position is a function of the free energy difference between reactants and products.
General properties of enzymes
Specificity, catalysis, regulation.
Enzymes have a pH optima. EVERY ONE DOES. Majority of them have a pH optima of around 7 (physiological).
Specificity is controlled by structure - the unique fit of substrate with an enzyme controls the selectivity for substrate and the product yield.
Active site can contain amino acids from different positions in the linear sequence of amino acids.
Enzymes are stereospecific. It matters where a functional group is in a structure. Noncovalent interactions stabilize substrate binding.
Importance of binding energy
Interaction between enzyme and substrate through multiple noncovalent interactions results in the release of free energy - this is the binding energy. When a substrate binds to an enzyme, in the active site, there is a release of free energy. Specificity results when the binding energy is maximized!!
Formation of the transition state; decreasing activation energy
Substrate binds to enzyme, noncovalent interactions are formed. Maximal binding energy release with the formation of the transition state; promotes catalysis.
Enzymes work by decreasing activation energy - by binding the transition state of a reaction better than the substrate. The transition state has a higher free energy than reactants or products because the bonds are strained. The transition state is stabilized by forming bonds with enzyme - enzymes facilitate the formation of a transition state - this decreases the activation energy.
Multiple weak noncovalent interactions between enzyme and substrate as a driving force.
Determines the rate of the reaction (velocity)
Net energy change
Determines the equilibrium
Functional properties of enzymes
-Accelerates forward and reverse reactions (depending on the equilibrium)
Decreases the activation energy for a reaction
Do NOT alter ΔG°' or Keq
-Accelerate the attainment of equilibria but do not shift the position. Equilibrium position is a function of the free energy difference between reactants and products.
Shows the relationship between substrate concentration and reaction rate (velocity)
Michaelis Menten - Background
k1 is the rate of formation of ES
k2 is the rate of dissociation of ES to E+S
k3 is the rate of conversion of ES to E+P
Assumptions: Formation of an enzyme-substrate complex, the ES complex is in rapid equilibrium with free enzyme, breakdown of ES to form products is assumed to be slower than formation of ES and breakdown of ES to re-form E and S.
The MM equation is given by
Vi=(Vmax[S])/(Km + [S])
Vi=Vo (initial velocity)
Vmax = maximal velocity
ET: total enzyme concentration
Vmax = k3[ET]
Km is related to the rate constants of the individual steps in catalysis.
Km is also a measure of the strength of the ES complex.
Y-axis: reaction velocity (rate; umoles product formed per unit time)
When [S] <<Km, V is proportional to [S]
=> First order kinetics, rate depends on concentration of substrate.
When [S] >> Km, V=Vmax
-Enzyme is saturated
V is independent of [S] => (Zero order kinetics, rate does not depend on concentration of substrate)
MM plot gives a hyperbolic plot for V vs [S]
Problem: Requires extrapolation of asymptotes
Solution: Linear transformation of the equation (lineweaver-burk, hanes-woolf)
Km is a constant -- a property of an enzyme for its substrate
Km is the substrate concentration for which the velocity is 1/2 Vmax
Km is an estimate of the dissociation constant of E from S
If an enzyme binds more than one substrate, the enzyme will have a distinct Km for each substrate.
Low Km = Tight Binding
-Advantageous in the cell: Limited [S], Solubility, Osmotic effects.
-Hexokinase is low Km
High Km = Weak Binding
-Effective at high [s]
-Glucokinase is high Km
Theoretical maximal velocity
Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality
To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate
Vmax is asymptotically approached as substrate is increased. At substrate concentration above the Km, zero order kinetics => V is no longer dependent on substrate concentration. At or below Km, Vmax follows first-order kinetics - Vmax is dependent on/proportional to the substrate concentration
Turnover number (kcat)
kcat = Vmax/[Et]
Turnover number = moles of substrate are converted to product per mole of enzyme (at saturation - vmax) per sec
Tells us how rapidly enzyme works.
Kcat/Km: measure of catalytic efficiency
Looking at different potential substrates, dividing Kcat by Km gives a measure of catalytic efficiency
Most enzymes in the cell are not saturated with substrate.
Considers rate of catalysis with a particular substrate
Strength of enzyme -substrate interaction
Double reciprocal plot
Very useful for predicting effects of inhibitors on an enzyme's activity
X-Intercept = -1/Km
Y-Intercepy = 1/Vmax
Slope = Km/Vmax
Don't disable enzyme forever.
Characterized by rapid dissociation of enzyme-inhibitor complex
Types: competitive, noncompetitive, uncompetitive
Irreversible inhibitors are either:
-covalently bound to the enzyme (bond wont break easily)
-noncovalently bound and very slow dissociation (tight, noncovalent binding; reduces the active concentration of the enzyme; in order to catalyze the reaction, the enzyme must be resynthesized)
-sucide inhibitors (enzyme is completely inactive once it is bound covalently to the active site by this compound)
Generally involve covalnt bond formation
Distinguished by a time-dependent decrease in enzyme activity
Group specific - react with R groups of amino acids
Suicide inhibitors - substrate analogue leads to enzyme inactivation
Penecillin - Transition state analog (suicide inhibitor for the enzyme involved in bacterial wall synthesis)
Binds to active site.
Structurally similar to substrate
Overcome by increasing [s]
MM plot - all reach the same Vmax, but more [s] is needed
Inhibitor will bind to some other site on the enzyme causing a conformational change. Substrate can still bind, but catalysis wont be as productive.
Vmax is decreased.
Inhibitor lowers concentration of functional enzyme.
MM plot: Km is the same, but Vmax decreases
Inhibitor is binding to the ES complex.
Lineweaver-Burk plot shows Vmax is decreased, Km is also decreased.
Km decreases because ES complex is removed.
Can distinguish it from noncompetitive because there are two parellelt lines in uncompetitive that dont intersect.
[Enzyme] has been lowered and stuck in the ES complex, cannot go any further to product. Enzyme is tied up, means inhibitor is binding to the ES complex, wont be able to reach VMAX.
Do NOT show Michaelis-Menten kinetics
Characterized by SIGMOIDAL kinetics
-Show cooperativity (subunits communicate with each other via noncovalent interactions)
-Hemoglobin shows allosteric behavior even though it is not an enzyme
Conformational change usually involves binding small effector molecules that are not substrates - these bind to an ALLOSTERIC site on the enzyme that results in rapid changes in enzyme activity
Allosteric Behavior: an important characteristic of regulatory enzymes
Respond to environmental signals - adaptive response
Small changes in allosteric modifiers can result in large changes in activity
This type of regulation may explain why enzymes are much larger than would be expected based on catalysis alone.
Catalyze the committed (rate-limiting) step in biochemical pathways
Allosteric Behavior: Concerted Model
Premise of concerted model
-All subunits exist as either T or R
-Substrate binds more readily to R
-Multiple active sites on different polypeptide chains
Binding of S disrupts T <-> R equilibrium in favor of R => cooperativity
Accounts for sharp increase in Vo in plot of V vs [S]
Regulatory molecules alter the T <-> R equilibrium
Positive effectors stabilize the R state
Negative effectors stabilize the T state
Positive effectors - left shift
Negative effectors - right shift
Allosteric Behavior: Sequential Model
Sequential model: subunits of allosteric enzyme undergo sequential changes in conformation on binding substrate. Binding of substrate to one subunit favors conformational change in adjacent subunit. Accommodates negative cooperativity.
Change in conformation is much more gradual
Shift in equilibrium between K1 and K2
Eventually shifts to the R conformation
Can account for negative cooperativity - sometimes an allosteric modifier could bind to an enzyme and actually reduce its sensitivity for substrate concentration
Enzyme Regulation: Phosphorylation/Dephosphorylation
In enzyme regulation: Kinases add phosphates to AAs w/ hydroxyl groups (serine, threonine, tyrosine) using ATP as a substrate.
Changes in phosphorylation/dephosphorylation are initiated by some external signal (for example, a hormone or neurotransmitter)
Phosphorylation/Dephosphorylation rapidly alters the activity of an enzyme
+addition of a phosphate to a protein
-introduces negative charges
-disrupt or form electrostatic interactions
-can form hydrogen bonds
+addition of a phosphate to a protein is a covalent bond
-reversible through the action of phosphatases
+activity may be increased or decreased - effect is specific to the enzyme
Enzyme Regulation: Acetylation
Adding 2 carbons (acetate) to amino terminus or side chain of Lysine/Arginine
Ex: Nucleosome structure - DNA + histones (small, basic proteins made of mostly arginine and lysine). Acetylation of histones neutralize the charge, unwraps DNA for transcription. DNA backbone = negative charge and histones = positive charge, electrostatic interactions holding the thing together
Acetylation is a way to neutralize charge on histone so interaction w/DNA is weakened so DNA can be unwrapped and other protein complexes can have access to it.
Enzyme Regulation: ADP Ribosylation
Adding ribose to N of arginine, glutamine; S of cysteine
Enzyme Regulation: Zymogen Activation
Zymogen activation is irreversible conversion of an inactive precursor to active enzyme by cleavage of covalent bonds
Enzyme Regulation: Modifications that determine intracellular/extracellular location and activity: Lipid addition
LIPID ADDITION- to cysteines and glycines
-Adding a hydrophobic tail to protein to allow it to insert in a membrane or be brought near a membrane where it needs to act
-modifying soluble proteins by addition of fatty acid to carry out specific function
-affects location and proximity
• Palmitoylation: internal SH of cys
• Myristoylation: NH of N-terminal gly
• Prenylation: SH of cys (like binds 2 cys together by their SH)
Enzyme Regulation: Modifications that determine intracellular/extracellular location and activity: Carbohydrate addition
CARBOHYDRATE ADDITION - to serine, threonine, or tyrosine, [or asparagine?]
-Providing a molecule with its own GPS device
-Type of sugar added will direct protein to intracellular compartment or plasma membrane
-----All proteins that face EC will have carbohydrate addition
-----Blood typing is based on carbohydrate residues present on surface of cell
-Directs protein to where it needs to be, primarily intracellular destination
• O-glycosylation: OH of ser, thr, tyr
• N-glycosylation: NH2 of asn
Mechanisms of Enzyme Regulation
-fundamental to all enzymes - no substrate, no rxn
-common to all enzymes
-cons: takes time to raise substrate concentration, osmotic effects
Amount of enzyme
-depends on synthesis + degredation
-both processes rgulate amount/activity, but are slow processes overall
-activators/inhibitors; feedback inhibition
Reversible covalent modification
-phosphorylation (fairly rapid process) and other types
-larger protein molecule is selectively cleaved to produce active form of enzyme (slow process)
Enzyme Regulation: Allosteric Enzymes
Only produce what cell needs, which is why enzymes are not active all the time
Positive and negative modifiers bind to allosteric sites
As concentration of allosteric modifiers builds up, odds that modifier will be bound is greater
• affects R -> T equilibrium
Feedback inhibition is a good mechanism to ensure you only produce what is needed. Don't waste additional substrate or energy.