Biochem Quiz #2
two properties that are unique to allosteric enzymes: (
(1) regulation of catalytic activity and (2) sigmoidal kinetics.
Vmax
maximal velocity of an enzyme at saturation conditions, high substrate concentration
The concerted model for allosteric enzymes
(A) [T] >>> [R], meaning that L0 is large. Consequently, it will be difficult for S to bind to an R form of the enzyme. (B) As the concentration of S increases, it will bind to one of the active sites on R, trapping all of the other active sites in the R state (by the symmetry rule.) (C) As more active sites are trapped in the R state, it becomes easier for S to bind to the R state. (D) The binding of S to R becomes easier yet as more of the enzyme is in the R form. In a velocity-versus-[S] curve, V0 will be seen to rise rapidly toward Vmax. The activity of allosteric enzymes is more sensitive to changes in substrate concentration near KM than are Michaelis-Menten enzymes with the same Vmax.
equation of an enzyme catalyzed rxn
1-substrate binding 2-catalytic step IGNORE REVERSE REACTION OF Product FORMING Substrate
Lineweaver-burk (Y=mx+b)
1/V0= Km/Vmax(1/[S])+1/Vmax
Regulator Molecules Modulate the T and R Equilibrium
A positive effector binds to the R form at a regulatory site, distinct from the active site, and stabilizes this form, thus increasing the concentration of R and making an R and S interaction more likely. A negative effector binds to T and stabilizes it; a negative effector increases the concentration of T and thereby decreases the likeli- hood of an R binding to an S The positive effector lowers the threshold concentration of substrate needed for activity, whereas the negative effector raises the threshold concentration.
The effect of regulators on the allosteric enzyme aspartate transcarbamoylase (ATP).
ATP is an allosteric activator of aspartate transcarbamoylase because it stabilizes the R state, making it easier for substrate to bind. As a result, the curve is shifted to the left, as shown in blue. Cytidine triphosphate (CTP) stabilizes the T state of aspartate transcarbamoylase, making it more difficult for substrate binding to convert the enzyme into the R state. As a result, the curve is shifted to the right, as shown in red.
allosteric enzymes
Act as information sensors• Are regulated by products of the pathways under their control
Allosteric enzymes and the committed step of a metabolic pathway
Allosteric enzymes always catalyze the committed step of metabolic pathways. allosteric enzymes can recognize inhibitor molecules as well as stimulatory molecules.
How do Allosteric enzyme kinetics differ from Michaelis-Mitten
Allosteric enzymes are distinguished by their response to changes in substrate concentration in addition to their susceptibility to regulation by other molecules. A typical velocity-versus-substrate curve for an allosteric enzyme is shown The curve differs from what is expected of an enzyme that conforms to Michaelis-Menten kinetics because of the sharp increase in V0 in the middle of the curve. The observed curve is referred to as sigmoidal because it resembles the letter S.
allosteric enzymes and metabolic pathways
As is typical in real metabolic situations, the end product F is needed in limited amounts and cannot be stored. The initial reactant A is valuable and should be conserved unless F is needed. Finally, B, C, D, and E have no biological roles except as chemical intermediates in the synthesis of F. This last condition means that the first reaction, A→ B, is the committed step.
Sulfanilamide: use and type of inhibitor
Humans do not have dihydropteroate synthetase. THF in humans is synthesized from dihydrofolate (a derivative of folic acid obtained via diet). However, some bacteria cannot take up folic acid and thus rely on de novo synthesis THF via dihydropteroate synthetase -sulfanilamide kills bacteria in humans
How can the production of F be regulated to meet the cellular requirements without making more than is needed?
In the simplest situation, when sufficient F is present, F can bind reversibly to e1, the enzyme catalyzing the committed step, and inhibit the reaction, an effect called feedback inhibition.
what is kcat
Kcat=turnover # of K2 the number of substrate molecule each enzyme site converts to product per unit time.
Michaelis-Menton Constant and equation
Km is the Michaelis-Menten constant which shows the concentration of the substrate when the reaction velocity is equal to one half of the maximal velocity for the reaction. Km=(K-1+K2)/K1
What happens when Vo = Vmax/2?
Km=[S]
Glyphosate (Roundup)
Product used in agriculture that blocks an enzyme (EPSPS) required for photosynthesis Glyphosate functions by occupying the binding site mimicking an intermediate state of the enzyme substrates complex.
The Sequential Model of Allosteric Enzymes
The binding of a substrate (S) changes the conformation of the subunit to which it binds. This conformational change induces changes in neighboring subunits of the allosteric enzyme that increase their affinity for the substrate. K1, K2, etc., are rate constants for the binding of substrate to the different states of the enzyme.
heterotropic vs. homotropic effects
The effects of regulatory mol- ecules on allosteric enzymes are referred to as heterotropic effects. Heterotropic effectors shift the sigmoidal curve to the left (activators) or right (inhibitors). In contrast, the effects of substrates on allosteric enzymes are referred to as homotropic effects. Homotropic effects account for the sigmoidal nature of the kinetics curve.
Mixed Inhibitor
The inhibitory molecule binds to separate (allosteric) site on the enzyme. Thus, the inhibitor can bind to either the E and ES complex.Binds to the enzyme at an allosteric site; inhibits product formation and does affect the affinity of the enzyme for the substrate.
Competitive Inhibition and rate of catalysis
The inhibitory molecule resembles the substrate and binds to the same active site of the enzyme. A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate; the inhibitor "competes" with the substrate for the active site.
What is catalytic efficiency
Together they show an enzymes preference for different substrates. kcat/KM results in the rate constant that measures catalytic efficiency.
Uncompetitive Inhibition
Uncompetitive Inhibitor - the inhibitory molecule binds only to the enzyme-substrate (ES) complex. Binding of the substrate to the enzyme produces the binding site for the uncompetitive inhibitor to bind to the ES complex.
What happens at low substrate concentrations to v0?
V0=(Vmax/Km)[S] Slope is relatively constant
what is the V0, and Vmax equation, and [ES]
V0=K2[ES] V0=(Vmax[S])/(Km+[S]) Vmax=K2[Et] one substrate binding enzyme
What happens at high substrate concentration
V0=Vmax slope is parabolic
Non-competitive Inhibition (vmax vs. km) and lineweaver-burke
Vmax-decreases Km-noeffect
Competitive inhibition on Vmax and Km (Michaelis Menton plot and Lineweaver-Burk)
Vmax: No effect Km-increases
Mixed inhibitor and vmax vs. Km
Vmax: decerases Km: depends (a) When the inhibitor favors binding the free enzyme over the enzyme substrate complex. The KMfor the substrate increases. Similar to the effect of a competitive inhibitor. This is the most common type of mixed-noncompetitive inhibitor. (b) When the inhibitor favors binding to the enzyme-substrate complex over the free enzyme. The KM for the substrate decreases. Similar to the effect of an uncompetitive inhibitor.This type of inhibitor is very rare ("theoretical"
Uncompetitive inhibition and Vmax vs. Km Michaelis-Menton vs. Lineweaver-Burke
Vmax: decreases Km: decreases
The Steady State Assumption and equation
[ES] is constant k1[E][S]=k-1[ES]+K2[ES] concentration of the free enzyme and bound enzyme are equivalent "produced at the same rate it is consumed"
Feedback Inhibitor and binding
usually bear no structural resemblance to the substrate or the product of the enzyme that they inhibit. feedback inhibitors do not bind at the active site but rather at a distinct regulatory site on the allosteric enzyme.
what is kinetics (specifically enzyme kinetics)
• Study of the rate of chemical reactions • Enzyme kinetics is the study of the rate of enzyme-catalyzed reactions