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OBJECTIVES
This lab is to learn the principles and fundamentals of enzyme kinetics. Alkaline
phosphatase will be used in this lab. Place all tables and graphs in you log book. These
must be completed and available for the TAs to look a t when the log books are graded.
BACKGROUND
Kinetics concerns the study of reaction rate and the conditions which affect reaction
velocity. The rates of enzymatic reactions are affected by various environmental factors
such as (a) pH and temperature, (b) the concentration of the enzyme, (c) the
concentration of the substrate, and (d) the presence and concentration of enzyme
activators, modifiers and inhibitors.
AP
R-monophosphate + H2O ------>ROH + orthophosphate
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Substrate specificity, relative rates and Km:
• Alkaline phosphatase (AP) hydrolyzes phosphate esters of primary and secondary
alcohols, cyclic aliphatic alcohols, sugar alcohols, phenols and amines.
• AP also hydrolyzes inorganic pyrophosphate (at a pH optimum of approx. 8)
• 5'-terminal phosphates of single and double-stranded DNA or RNA.
• The enzyme will not hydrolyze phosphodiesters (R-O-PO2-O-R'; R, R'=alkyl
groups).
• Representative Michaelis constants (Km determined in 0.1 M Tris, pH 8.0 at
25oC) include
o 4-nitrophenyl phosphate, 3.6 µM (relative rate = 1.0);
o pyrophosphate, 16µM (rate = 0.9);
o AMP, 18µM (rate = 1.2)
o ATP, 16µM (rate=0.9)
o phosphoenolpyruvate, 5.5 µM (rate= 0.8).
• Note: The hydrolysis rate and substrate affinity of AP depends on a complex
inter-relationship between the purity and concentration of the enzyme, buffer
composition, ionic environment (ionic strength, solvents present) and pH.
• Specific uses for AP require careful empirical optimization of reaction conditions.
• Enzyme structure and Mr:
o AP is a dimer (Mr = 140,000) of identical or nearly identical subunits M r=
69,000), each of which contains 2 molecules of Zn2+, one tightly bound
and necessary for structural stability, the other loosely bound and required
for catalytic activity.
o The active site contains a reactive serine.
o In some mammals (e.g., humans), there are at least 3 distinguishable
isoenzymes: the intestinal, the placental and the form found in
bone/liver/kidney.
• pH optimum: 8.0-10.5 (depending on substrate concentration). The pH optimum
under quality control assay conditions is 9.8 (Under these assay conditions,
analytical grade AP has approx. 75% maximum activity at pH 9.0 and approx.
50% at pH 11.0)
• Activators:
o divalent metal ions (Mg2+, Co2+, Mn2+),
o amino alcohols (2-amino-2-methyl-1-propanol, diethanolamine),
o Tris buffer.
o Also, a low level of Zn2+ is required for enzyme.
• Inhibitors:
o inorganic phosphate (Pi),
o monoethanolamine, Be2+,
o chelators of divalent metal ions (EDTA, oxalate, citrate, cysteine,
histidine),
o acid or neutral pH,
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o aromatic amino acids (Phe, Trp),
o L-homoarginine,
o urea,
o iodoacetamide.
o High levels of Zn2+ are inhibitory.
• pl: 5.7.
• Absorbance of purified enzyme: 7.6 (10 mg AP/ml; 278 nm).
EXPERIMENTS
0.005
0.01
0.02
Graph the absorbance vs concentration and determine the E1M for p-nitrophenol from the
slope of the graph. Include this in your assignment
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3) Alkaline phosphatase: 3.3 µ g/ml in TRIS buffer
Mix substrate and buffer in Spectronic 20 tubes. Place tube one in the spectrophotometer
and zero the instrument. Start the reaction, one tube at a time, by adding enzyme.
Immediately after addition of the enzyme, mix thoroughly and transfer the solutions to
the Spectronic 20.
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Table 3. Class results for the effect of increasing enzyme concentration.
Method
Tube number 1 2 3 4 5
Final substrate
concentration
Substrate (stock 0.025 0.05 0.075 0.1 0.2
solution*) ml
0.1M Tris-HCl,
pH 8.0 (ml)
* Concentration of stock substrate solution: 1 mg/ml
Note: substrate is in Tris-HCl, pH 8, buffer
• The final incubation volume, after the addition of enzyme, will be 3 ml.
b) Zero each tube individually. Working with 1 tube at a time, add 0.15 ml of
enzyme (3.3 ug/ml) and measure the increase in absorption at 30 second intervals
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for the first 3 minutes and at 1 minute intervals for the next 2 minutes. Record the
results in Table 5.
Plot a graph of 1/v vs 1/[S] using your results and the means of the class results.
Determine the values of Vmax and Km. Hand this in with your assignment.
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Table 5. The effect of substrate concentration on enzyme activity. (Group results)
0.5
1.5
2.5
Mean
∆ A/min.
µ mol p-
nitrophenol
produced/min
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Table 6. Class results for the effect of substrate concentration.
Mean +/-
St.Dev.
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4.The Effect Of A Metal-Chelating Agent, EDTA, On Alkaline Phosphatase Activity
All solutions for use in this experiment are cation-free. Prepare three test tubes as
follows:
TUBE A TUBE B
a) substrate: pNPP 1 mg/ml (ml) .075 .075
b) 0.2 M EDTA (ml) - 0.015
c) TRIS-HCl buffer
Note: EDTA = Ethylene diamine tetraacetic acid (What is the final concentration of
EDTA in the assay?); substrate is in Tris-HCl buffer, pH 8.0. pH of EDTA is
adjusted to pH 7.0.
Add sufficient TRIS buffer so that the final volume will be 3 ml after the addition of
enzyme. Zero each tube and then add 0.15 ml of enzyme (3.3ug/ml) to start the reaction.
Immediately after the addition of enzyme, mix thoroughly and place tube back in the
spectrophotometer. If time and enzyme permit repeat tube B using twice as much EDTA.
Measure the increase in absorption in tubes A and B at 405 nm at 1 minute
intervals for 5 or 6 minutes (Table 7). Plot absorbance vs time for each condition on
the same graph. Calculate the initial velocity of the enzyme reaction in terms of
pmoles of p-nitrophenol liberated per minute and compare results of class data in
Table 8. What is the effect of EDTA? Hand in this graph and answer the question
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Table 7. The effect of EDTA on alkaline phosphatase activity. (Group results)
vo: ∆ A/min.
vo: µ mol p-
nitrophenol
produced/min
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Table 8. Class results for the effect of EDTA on alkaline phosphatase activity.
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Assignment:
Hand in the following in the week of Feb 14, 2011. Maximum 2 double spaced pages
not including graphs/tables and references:
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