Laboratory exercise 2

Week of Jan 24, 2011

ENZYME KINETICS: ALKALINE PHOSPHATASE

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.

Enzyme catalyzed reactions can be followed by monitoring either the rate of

disappearance of the substrate or the rate of appearance of the product. It is generally
preferable to assay newly formed product since this can be easily measured compared to
accurately measure the loss of a small proportion of the supplied substrate. For some
enzymes, they do not have absolute substrate specificity. In this case, the characteristics
of the enzyme can be analyzed by using a synthetic substrate which is readily assayed
spectrophotometrically. Such an assay using an “artificial” substrate can be
demonstrated in the case of alkaline phosphatase.

Alkaline phosphatase is widely distributed in animal tissue and is an orthophosphoric

monoester phosphohydrolase.

AP
R-monophosphate + H2O ------>ROH + orthophosphate

This enzyme can be assayed by measuring the liberation of orthophosphate from a

suitable monophosphate ester. However, a more convenient method is to use p-
nitrophenyl disodium orthophosphate as the substrate.
• Enzymatic hydrolysis at an alkaline pH yields the yellow anion of p-nitrophenol
which can be measured spectrophotometrically.
• In alkaline solution, p-nitrophenol absorbs at 405 nm.
• The substrate p-nitrophenyl phosphate does not absorb at this wavelength. So the
progress of the enzymatic reaction can be readily followed by measuring the
change in extinction at 405 nm.

Properties of Alkaline Phosphatase

(Boerhinger Mannheim, Biochemica Information, 1987. Boerhinger Mannheim is now
part of Roche)

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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

1. Standard Curve For p-Nitrophenol

Plot a graph of extinction (405nm) against concentration using a range of p-nitrophenol
solutions prepared from the standard solution. This will allow you to determine the E1M
for p-nitrophenol. Dilute the 0.05 mM standard solution of p-nitrophenol with alkaline
buffer to give 3 ml each of 0.005, 0.01, and 0.02 mM p-nitrophenol. Measure the
absorbance of each solution at 405 nm using the alkaline buffer to zero the
spectrophotometer.

Standard Curve For p-Nitrophenol

p-Nitrophenol Concentration Absorbance (405 nm)

(mM)

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

2.Variation Of Alkaline Phosphatase Activity With Enzyme Concentration

Prepare 5 test tubes as follows:

Tube Number
Additions 1 2 3 4 5
substrate1 (ml) 2.0 2.0 2.0 2.0 2.0
Buffer2 (ml) 1.0 0.9 0.8 0.7 0.6
Alkaline phosphatase3 (ml) - 0.1 0.2 0.3 0.4

1) p-nitrophenyl phosphate: 40 µ g/ml (Mr of pNPP = 371)

2) buffer: 0.1 M Tris-HCl, pH 8.0

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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.

Measure the increase in extinction or absorption at 405 nm in each tube at 30 second

intervals for the first 3 minutes and at 1 minute intervals for the next 2 minutes. Plot
the absorbance values vs time for each of the enzyme concentrations on a single graph.
Calculate the initial velocity for each concentration of enzyme in terms of pmoles p-
nitrophenol liberated per minute. Plot this activity against enzyme concentration using
your own data (Table 2) and the means from the class data (Table 3).

Table 2 Effect of enzyme concentration on rate of hydrolysis of p-nitrophenyl

phosphate. (Group results)

Absorbance (405 nm)

Enzyme vol. 0.1 ml 0.2 ml 0.3 ml 0.4 ml
Time (min)
1 min
1.5 min
2 min
2.5 min
3 min
4 min
5 min
mean ∆ A/min
vo: µ mol
p-nitrophenol
liberated/ min
* Record values at 1 decimal place only.

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Table 3. Class results for the effect of increasing enzyme concentration.

Initial velocity (µ mol p-nitrophenol/min x 10-3)*

Group 0.1 ml 0.2 ml 0.3 ml 0.4 ml
1
2
3
4
5
6
7
means +/- St
Deviation
* Record values at 1 decimal place only.

Plot product production versus time for the enzyme concentrations

Plot average (with standard deviations) of the initial velocity versus enzyme
concentration, include this in your assignment

3. The Effect Of Substrate Concentration On Enzyme Activity

Method

a) Prepare 5 test tubes to contain a final volume of 2.85 ml of p-nitrophenyl

phosphate at varying concentrations as in Table 4 (Use the Tris buffer to adjust).

Table 4. The effect of substrate concentration on the activity of alkaline

phosphatase.

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.

c) Calculate the initial velocity at each concentration of substrate in terms of

pmoles of p-nitrophenol liberated per minute.

d) Plot a graph of product production versus time at different substrate

concentrations Plot a graph of velocity versus substrate concentration using your
own results and the means from the class results (Table 6).This will be handed in
as part of your assignment

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)

Absorbance (405 nm)

[S]
Time (min)

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.

Initial velocity (mmol/min)

[S]
Group

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)

Absorbance (405 nm)

- EDTA + EDTA
Time (min)

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.

Initial velocity (mmol/min)

- EDTA + EDTA
Group

Mean +/- St.

Dev

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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:

1. standard curve for p-nitrophenol with extinction coefficient

2. Plot average (with standard deviations) of the initial velocity versus enzyme
concentration, include this in your assignment
3. velocity versus substrate concentration using your own results and the means from
the class results (Table 6).
4. 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. Also plot this data as a Hanes-Woolf plot
5. Effects of EDTA: absorbance versus time for each condition (i.e. +/- EDTA).
Calculated the initials velocity of each reaction and briefly compare this to the
group results. What is the effect of EDTA?
• Hand in a brief introduction (one paragraph ) along with the above
graphs and the related tables, calculations , appropriate legends and data
analysis (no more than 2 pages) in the week of Feb 14, 2011.

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