Presentation on theme: "LAB 3 Enzyme Kinetics Studying -galactosidase activity at varying substrate concentrations in the presence and absence of an inhibitor Michaelis-Menten."— Presentation transcript:
1LAB 3 Enzyme KineticsStudying -galactosidase activity at varying substrate concentrations in the presence and absence of an inhibitorMichaelis-Menten enzyme kineticsLineweaver Burk plot
2Michaelis-Menten Enzyme Kinetics Steady-state Model:Formation of ES = Disappearance of ESk1[E][S] = k2[ES] + kcat[ES]Define a new constant, Km (Michaelis constant):Km = (k2 + kcat)/k1Km reflects the affinity of the enzyme for the substrate:• low Km = high affinity• Km = [S] that produces V= 1/2Vmax
3The Michaelis-Menten Equation V =Vmax[S]Km + [S]Hyperbolic function1/2Vmax
4Simplifying M & M analysis using a Lineweaver-Burk Plot • A double-reciprocal representation of V vs. [S]data: plot 1/V vs. 1/[S]• Take the inverse of both sides of the M-Mequation to get the L-B equation, whichspecifies a line.V =Vmax[S]Km + [S]M-MInverse M-M1/V = (Km + [S]) / Vmax[S]Rearrange…L-B1/V = 1/Vmax + Km/ Vmax (1/[S])
5Determining Kinetic Parameters from an L-B Plot 1/V = 1/Vmax + Km/ Vmax (1/[S])
6Last week’s experiments vs. today’s Last week studied enzyme at saturating substrate concentrations, meaning there was so much substrate that the enzyme worked at Vmax all the time so we could determine specific activity.Today, varying concentration of substrate to find out what happens when substrate is limiting. How strong of an affinity does the enzyme have for this substrate? Can determine the Michaelis constant, Km.
7TO DO TODAYVary substrate (ONPG) conc. over an 80-fold range to determine Km, the substrate conc. that gives V = 1/2 V maxRepeat above using a constant amount of an inhibitor (IPTG) to determine Km in the presence of the inhibitor.Determine whether the inhibition is competitive or non-competitive by comparing Km & Vmax -/+ IPTG on Michaelis-Menten & Lineweaver-Burk plots.
8Competitive Inhibition No product formedInhibitor has a chemical structure similar to that of the substrate and competes for binding to the active site (can only bind free E).Inhibition can be overcome by excess substrate.Inhibitor increases Km but leaves Vmaxunchanged.
9Noncompetitive Inhibition Diminished product formationInhibitor’s chemical structure may be totally different from that of the substrate.Inhibitor binds a site on the enzyme that is distinct from the active site.EI complex can bind substrate, but cannot transform it into product as efficiently as the uninhibited enzyme because of alterations in the shape/chemistry of the active site.Inhibition cannot be overcome by excess substrate.Inhibitor decreases Vmax but leaves Km unchanged.
12Vmax +I = Vmax -I / ( 1 + [I]/Ki) The dissociation constant for an inhibitorWhereas Km reflects the affinity of anenzyme for its substrate, Ki reflects itsaffinity for binding to an inhibitor.• Method of calculating Ki depends on thetype of inhibition.For competitive inhibition:Km +I = Km -I ( 1 + [I]/Ki)For noncompetitive inhibition:Vmax +I = Vmax -I / ( 1 + [I]/Ki)
13Two more types of inhibition 1. Mixed inhibitionInhibitor can bind both E and ES, but with different affinities (different Kis).Both Vmax and Km may change; if inhibitor binds E with higher affinity than ES, Vmax decreases and Km increases.2. Uncompetitive inhibition (quite rare)Inhibitor binds only ES (not free E).Both Vmax and Km decrease by same factor.
14Substrates & an inhibitor of b-galactosidase What kind of inhibitor is IPTG likely to be?
15TO DO TODAYAssay the dilution of PF of beta-gal from last week that gave Abs of approx. 0.5(Remember it has been diluted 2/3 with glycerol)DO NOT ADD ENZYME TO REAGENT BLANKS (TUBES #10 & 11)DO NOT add the enzyme until timing startsTime carefully, mix wellDo the assay twice, once without inhibitor and again with inhibitor (IPTG)Make Michaelis-Menten and Lineweaver -Burk plots using Excel following directions in appendix after Lab 3 in lab manualMake sure you understand homework calculations—15 points
16Homework Calculations 15 points Protein conc. (mg/ml) for PF using lab 2 result (adjusted for addition of glycerol)ONPG & IPTG conc. in nmol/mlVolume used = 2.4 mlStock ONPG (mol. wt 301.3) = 4 mg/mlStock IPTG (mol. wt 238.3) = 0.6 mg/mlVelocity (Spec. Activity)Units = nmol/min/mg proteinVolume used = 3.4 mlMichaelis-Menten plot (ONPG is substrate)without IPTGwith IPTGLineweaver-Burk plotKi, disassociation constant of Inhibitor