BIOCHEMISTRY LAB. CHE555. Lecture 6: 3 March 2011. Enzyme Kinetics. A
chromogenic reaction catalyzed by β-galactosidase is used to visualize the
effects ...
Background (Enzyme Kinetics)
BIOCHEMISTRY LAB CHE555 Lecture 6: 3 March 2011 !
Enzyme Kinetics A chromogenic reaction catalyzed by β-galactosidase is used to visualize the effects of substrate concentration and inhibitors of different types on enzyme-catalyzed reaction velocity. Experiment #7, pages 95-104, 123-130. days 1 and 2
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Enzymes influence the rate at which equilibrium is obtained, but not the equilibrium position of the reaction. Kinetics, in this context, is the study of reaction product formation as a function of time. The rate of product formation is a measure of the reaction velocity.
S
→
P
d([P]) d([S]) v= =− dt dt v = reaction velocity
Enzymes accelerate reactions by up to x 1021
Biology needs reactions that are not ‘naturally’ fast, because it needs stable compounds.
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Wolfenden and Snider (2001) Acc. Chem. Res. 34:938-945.
The reaction is accelerated by lowering the free energy of transition state activation. ‡ kvel = kBT e-ΔG /RT
h
[S]eq [P]eq
Keq = e-ΔG°/RT
time [P]0
d[P]/dt = v v0 =
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k3[ET] [S]0 KM + [S]0
We measure the initial reaction velocity, when [P] ≈ 0 and [S] ≈ [S]0.
ΔF° = ΔG° = -RT ln Keq
Background (Michaelis-Menton)
Observed Initial Rate of Reaction !
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measure and plot initial velocity (vo) as a function of substrate concentration ([S]0). Initial velocity is used to eliminate effects of product buildup (slowing down reaction). At some point adding more substrate does not further increase the reaction rate. A plateau is observed corresponding to saturation of the enzyme. For single substrate, Vmax occurs when all of substrate is in ES state.
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Figure II-8
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The Michaelis-Menton equation describes the relationship between initial velocity (vo) and initial substrate concentration ([S]0 ).
KM (the Michaelis constant) is equal to the substrate concentration that yields vo equal to Vmax/2 . This constant is used as a general measure of the stability of the enzyme substrate interaction. KM has the form of dissociation constant. The analogy is best when k3 28C B->A L28C B L25C A L25C B L38C A L38C B L44C A L44C B L41C A L41C B L35C A L35C B
Sam Beavin
L41C
Brittany Buurman
L35C
Sarah Garner Andrea Hiller Shuang Liu Sarah Negaard Brandon Nelson Richard Rathbun Jesse Reed Steven Shofner Joseph Bodnar Stephanie Carpenter John Drury Maks Gold Emily Hall Byron Hempel Susannah Hubler Matthew Hudzinski Jack Moore Leah Neeley Jessica Phelps Michael Sudkamp
We will follow the experimental protocol exactly for Days 1 and 2 except
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Skip steps 11-14 on Day 1 (determination of enzyme concentration) use either MGP, or MTG on Day 2 (see prev. pg. for who should do which). omit steps 11-13 on Day 2.
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We will not be doing Days 3 and 4. Technical tweaks – –
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Do not vortex enzyme-containing solutions. Invert or swish in and out of a pipettor. Start a timer as you add enzyme. Record the time at which you make your first absorbance reading if it is not exactly at 30 sec. Do the same for any time point that is not at the target time. Work with a buddy who can adjust your pipettor for the next addition while you work with the spectrophotometer. For step 10, first plot activity vs. enzyme amount present, and use only points that fall in a linear regime, when calculating your stock solution activity.
Data Analysis
Run your long time course with a buddy, each of you can put a tube in the same spectrophotometer and you can each take turns reading absorbances
Data Analysis Prepare a table in advance so that during the experiment you will be filling in boxes with absorbances (also allow a column for time of observation). Write your results down in real time.
Example Data from Day 1
Linear regime for enzyme activity
A420
For point 10, instead of averaging, use the slope of the linear range to calculate activity on a per-volume basis (volume of stock).
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Enzyme Activity at One Concentration
pe o l o s me t i ted vs. t a l re 20 A4
of
A420
related to 1/dilution
Look for linearity over 2-4 minutes
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Experimental Considerations Pay careful attention to which steps have experiments and which have data analysis. You can do the data analysis later.
Safety Considerations
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Make sure your data is saved, for example to the hard drive of one of the computers, in case your memory stick goes bad. !
Delete all old files from the spec. to free up more ‘experiments’ for use. !
It will be easy to confuse substances, so please be careful. !
