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:
LAB 3 Enzyme Kinetics Studying - galactosidase activity at varying substrate concentrations in the presence and absence of an inhibitor Michaelis-Menten enzyme kinetics Lineweaver Burk plot
Michaelis-Menten Enzyme Kinetics Steady-state Model: Formation of ES = Disappearance of ES k 1 [E][S] = k 2 [ES] + k cat [ES] Define a new constant, K m (Michaelis constant): K m = (k 2 + k cat )/k 1 K m reflects the affinity of the enzyme for the substrate: low K m = high affinity K m = [S] that produces V= 1/2V max
The Michaelis-Menten Equation V = V max [S] K m + [S] 1/2V max Hyperbolic function
Simplifying 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-M equation to get the L-B equation, which specifies a line. 1/V = (K m + [S]) / V max [S] V = V max [S] K m + [S] M-M 1/V = 1/V max + K m / V max (1/[S]) Inverse M-M Rearrange… L-B
Determining Kinetic Parameters from an L-B Plot 1/V = 1/V max + K m / V max (1/[S])
Last 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 V max 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, K m.
TO DO TODAY Vary substrate (ONPG) conc. over an 80-fold range to determine K m, the substrate conc. that gives V = 1/2 V max Repeat above using a constant amount of an inhibitor (IPTG) to determine K m in the presence of the inhibitor. Determine whether the inhibition is competitive or non- competitive by comparing K m & V max -/+ IPTG on Michaelis- Menten & Lineweaver-Burk plots.
Competitive Inhibition Inhibitor 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 K m but leaves V max unchanged. No product formed
Noncompetitive Inhibition Inhibitor’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 V max but leaves K m unchanged. Diminished product formation
Michaelis-Menten Kinetics with Inhibition
Lineweaver-Burk Plots with Inhibitors
K i The dissociation constant for an inhibitor Whereas K m reflects the affinity of an enzyme for its substrate, K i reflects its affinity for binding to an inhibitor. Method of calculating K i depends on the type of inhibition. For competitive inhibition: K m +I = K m -I ( 1 + [I]/K i ) For noncompetitive inhibition: V max +I = V max -I / ( 1 + [I]/K i )
Two more types of inhibition 1. Mixed inhibition 2. Uncompetitive inhibition (quite rare) Inhibitor can bind both E and ES, but with different affinities (different K i s). Both V max and K m may change; if inhibitor binds E with higher affinity than ES, V max decreases and K m increases. Inhibitor binds only ES (not free E). Both V max and K m decrease by same factor.
Substrates & an inhibitor of -galactosidase What kind of inhibitor is IPTG likely to be?
TO DO TODAY Assay 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 starts Time carefully, mix well Do 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 manual Make sure you understand homework calculations—15 points
Homework Calculations 15 points Protein conc. (mg/ml) for PF using lab 2 result (adjusted for addition of glycerol) ONPG & IPTG conc. in nmol/ml –Volume used = 2.4 ml –Stock ONPG (mol. wt 301.3) = 4 mg/ml –Stock IPTG (mol. wt 238.3) = 0.6 mg/ml Velocity (Spec. Activity) – Units = nmol/min/mg protein – Volume used = 3.4 ml Michaelis-Menten plot (ONPG is substrate) –without IPTG –with IPTG Lineweaver-Burk plot –without IPTG –with IPTG K i, disassociation constant of Inhibitor