LAB 3 Enzyme Kinetics

1
LAB 3Enzyme Kinetics

• Studying ?-galactosidase activity at varying
  substrate concentrations in the presence and
  absence of an inhibitor
• Michaelis-Menten enzyme kinetics
• Lineweaver Burk plot

2
Michaelis-Menten Enzyme Kinetics
Steady-state Model Formation of ES
Disappearance of ES k1ES k2ES
kcatES Define a new constant, Km (Michaelis
constant) Km (k2 kcat)/k1 Km reflects the
affinity of the enzyme for the substrate low
Km high affinity Km S that produces V
1/2Vmax
3
The Michaelis-Menten Equation
Hyperbolic function
1/2Vmax
4
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.
M-M
1/V (Km S) / VmaxS
Inverse M-M
Rearrange
L-B
1/V 1/Vmax Km/ Vmax (1/S)
5
Determining Kinetic Parameters from an L-B Plot
1/V 1/Vmax Km/ Vmax (1/S)
6
Last weeks experiments vs. todays

• 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.

7
TO DO TODAY

• Vary substrate (ONPG) conc. over an 80-fold range
  to determine Km, the substrate conc. that gives V
  1/2 V max
• Repeat 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.

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Competitive Inhibition
No product formed

• 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 Km but leaves Vmax
• unchanged.

9
Noncompetitive Inhibition
Diminished product formation

• Inhibitors 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.

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Michaelis-Menten Kinetics with Inhibition
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Lineweaver-Burk Plots with Inhibitors
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• Ki
• The dissociation constant for an inhibitor
• Whereas Km reflects the affinity of an
• enzyme for its substrate, Ki reflects its
• affinity for binding to an inhibitor.
• Method of calculating Ki depends on the
• type of inhibition.
• For competitive inhibition

Km I Km -I ( 1 I/Ki)
For noncompetitive inhibition
Vmax I Vmax -I / ( 1 I/Ki)
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Two more types of inhibition
1. Mixed inhibition

• Inhibitor 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.

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Substrates an inhibitor of b-galactosidase
What kind of inhibitor is IPTG likely to be?
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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 calculations15
  points

16
Homework Calculations15 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
• Ki, disassociation constant of Inhibitor