Download Experiment 7

Experiment 7
pH OPTIMUM OF AN ENZYME
pH affects the observed activity of enzymes by its effect on stability and on
rates of conversion of substrate to product. In general, enzymes are denatured at pH
values below 2-3 and above 10-11 and are most stable near pH 6. However, there
are exceptions. Pepsin is most stable at pH 3 and is rapidly inactivated at pH 7. The
pH for maximum stability does not necessarily correspond to the pH for maximum
activity. For example, bovine trypsin and bovine chymotrypsin are most stable at
pH 3 while they have maximum activity at pH 7.8.
The rate of conversion of substrate to product is pH dependent because pH
may affect binding of substrate or cofactors to the enzyme and it affects the
ionization state of essential catalytic groups - such as carboxyl, imidazole,
sulfhydryl - known to be a part of the active site. For example, if a carboxyl group
in the active site of the enzyme is active only in the ionized form, then the activity
of the enzyme will be decreased at low pH because
E COOH
Kion
+
E COO- + H
the protonated form is favored. For example, based on pKa of 4 the enzyme would
have 9, 50, and 91% of its maximum activity at pHs 3, 4 and 5, respectively.
The observed pH-activity curve is a function not only of the enzyme but also
of the reaction conditions and activity measurement procedures. In Fig. 7-1 the
rates of formation of product at several different pHs are shown. The solid lines are
experimental data while the dashed lines represent initial rates. Initial rates are
obtained by drawing tangents to the experimental lines such that the tangential lines
go through zero. Thus initial rates are obtained as near the beginning of the
reaction as possible. As shown in Fig. 7-1 the experimental rates and initial rates
are essentially identical at pH 4.5, 5.0 and 6.0 but the two begin to differ at pH 7.0
and at pH 8.0 and 8.5 the two are markedly different. The difference between the
two in this case is most likely due to instability of the enzyme at the higher pHs.
In Fig. 7-2 are plotted the pH-activity curves for this enzyme system (Fig. 71) as determined from initial rate data (open squares) and at a fixed interval of 6
min (filled circles). The difference between the two curves is quite marked above
pH 6. The correct pH-activity curve is that obtained from initial rate data.
1
Figure 7-1. Effect of pH on rate of product formation. The solid lines indicate
experimental data, the dashed lines initial rates.
Figure 7-2. pH-activity curves based partially on data of Fig. 11-1. The open squares
represent data from initial rates while the filled circles represent data determined at 6 min
of reaction times.
The pH-activity curve is often influenced by the nature and concentration of
the substrate. In Fig. 7-3, the relative activity-pH curves for two substrates are
shown. Curves similar to these are found in the case of chymotrypsin-catalyzed
2
hydrolysis of N-acetyl-L-tyrosinamide and N-acetyl-L-tyrosine ethyl ester.
Chymotrypsin-catalyzed hydrolysis involves at least three steps as shown.
O
E
OH + R
C
O
NH
R'
E
OH . R
C
NH
acylation
R'
E
O
O
C
+
R'NH2
R
deacylation
E OH
H2O
+
R
COOH
The first step is formation of the Michaelis-Menten adsorptive complex,
which involves non-covalent bonds. The acylation step involves covalent bond formation to an ester followed by deacylation to the free enzyme and the acid. In the
case of N-acetyl-L-tyrosinamide hydrolysis the rate-determining step is acylation
with a bell shaped pH-activity curve (open squares). With N-acetyl-L-tyrosine
ethyl ester hydrolysis, the rate-determining step is deacylation with a sigmoidal pHactivity curve (filled circles).
Fig. 7-4 shows the pH-activity curves for the hydrolysis of phosphocreatine,
ß-glycerophosphate and phenylphosphate by calf intestinal mucosa alkaline
phosphatase. Not only the shape of the curves but also the pH optima are substrate
dependent.
Even the nature of an essential cofactor can affect the pH-activity curve. pHactivity curves for hydrolysis of phenylphosphate by calf intestinal mucosa alkaline
phosphatase in the presence of 0.005 M MgCl2 and 0.001 M MnCl2, respectively,
are shown in Fig. 7-5. The pH optimum is ~8 in the presence of MnCl2 but ~9.4
in the presence of MgCl2.
Figure 7-3. pH-relative activity curves for two different substrates (N-acetyl-Ltyrosinamide, ______; N-acetyl tyrosine ethyl ester, - - - - ) which have different ratedetermining steps (see text).
3
Figure 7-4. Effect of type of substrate on pH-activity curves for calf intestinal mucosa
alkaline phosphatase catalyzed hydrolysis. Open squares, 0.015 M phosphocreatine; open
circles, 0.02 M ß-glycerophosphate; filled circles, 0.025 M phenylphosphate [R. K.
Morton, Biochem. J. 61, 232 (1955)].
In summary, pH-activity curves are essential for describing the activity of an
enzyme. However, the curves are a property of the total system and not just the
enzyme and they may be influenced by the assay conditions used.
REAGENTS:
• Crude milk alkaline phosphatase. Alkaline phosphatase is bound to the fat
globule membrane and can be solubilized by isobutanol treatment. In addition,
the isobutanol at 38°C precipitates the caseins thus giving some purification.
Place 90 mL of unpasteurized fresh milk into each of two 250 mL centrifuge
bottles. Add 45 mL of isobutanol to each. Warm with continuous stirring for 5
min at 38°C. During this time coagulation of the caseins should have occurred.
If not, add 0.40 mL of glacial acetic acid with continuous stirring to each bottle
and reheat. (Depending on the age and bacterial population of the milk from 0.1
to 1 mL acetic acid may be needed to lower the pH sufficiently to cause
precipitation of the caseins). Centrifuge for 20 min at 8,000 rpm in a refrigerated
Sorvall centrifuge. Siphon off the isobutanol layer. Carefully decant the
aqueous layer onto a Buchner funnel containing about 0.5 cm layer of hyflo
supercel on a Whatman No. 2 filter paper. Filter under low vacuum. Refilter the
solution through Whatman No. 2 paper in a conical funnel using gravity
filtration. Approximately 140 mL of fairly clear filtrate should be obtained.
4
• As an alternative to the milk alkaline phosphatase, bovine intestine alkaline
phosphatase may be used (Sigma P-7640, 23 units/mg). Prepare a stock solution
of 12.75 u/mL, diluted to 0.25 u/mL for use.
• Glycine buffers. Prepare stock solutions of 0.2 M glycine (MW = 75.0) and
0.25 M NaOH (MW = 40.0). From these stock solutions prepare the following
8 buffers.
No.
0.2 M glycine
0.25 M NaOH
Water
(mL)
1
2
3
4
5
6
7
8
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
(mL)
0.40
0.90
1.40
1.96
2.72
3.40
4.55
(mL)
4.60
4.10
3.60
3.04
2.28
1.60
0.45
Adjust pH to 12.0 with NaOH; make to 10.0 mL.
• p-Nitrophenol. 5.00 x 10- 5 M . Prepare fresh daily by dilution of a stock
solution containing 0.1391 gm p-nitrophenol in 100 mL which is 0.01 M
(molecular weight of p-nitrophenol is 139.1).
• Disodium p-nitrophenyl phosphate. 2.50 x 10-3 M (molecular weight of
substrate is 264).
• NaOH. 0.2 M solution.
Figure 7-5. Effect of nature of activating cation on pH-activity curve for calf intestinal
mucosa alkaline phosphatase-catalyzed hydrolysis of 2.5 x 10-4 M phenyl phosphate.
5
Filled circles represent the use of MnCl2 as a cofactor; open squares represent the use of
MgCl2. [R. K. Morton, Biochem. J. 65, 674 (1957)].
6
PROCEDURE:
Disodium p-nitrophenyl phosphate will be used as the substrate and the
reaction will be followed in a Shimadzu spectrophotometer by the rate of increase
in absorbance at 403 nm due to formation of nitrophenol (see below).
Standard curve. In order to relate absorbance at 403 nm to concentration of
p-nitrophenol produced it is necessary to have a standard curve relating these two
parameters.
(colorless)
O
O2N
O
P
O + H2O
=
O2N
OH + HPO4
O
pKa 7.16
O
O2N
O2N
O
(yellow)
To prepare the standard curve, mix water, 5 x 10-5 M p-nitrophenol and 0.2
M NaOH in the amounts given.
Tube No.
1
2
3
4
5
6
7
p-Nitrophenol
Water
0.2 M NaOH
10-5
(5 x
M)
(mL)
(mL)
(mL)
0
0.5
1.0
2.0
3.0
4.0
4.5
4.5
4.0
3.5
2.5
1.5
0.5
0
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Mix thoroughly and transfer to a colorimeter tube. Read in the
spectrophotometer at 403 nm using tube no. 1 as blank.
7
pH optimum of alkaline phosphatase. To a test tube add 2.0 mL of one of
the glycine buffers, 2.0 mL of 2.5 x 10-3 M disodium p-nitrophenyl phosphate
solution and 0.80 mL water. Equilibrate in the bath or thermostated
spectrophotometer for a minimum of 5 min to bring to 37°C, remove the tube and
add 0.20 mL of enzyme, mix and rapidly return it to the spectrophotometer. Record
absorbance for 5 min at 20-second intervals or until the absorbance change is 0.3.
Immediately on completion of the reaction determine the pH of the solution.
Repeat for the other 7 buffers.
QUESTIONS:
1. What is the Beer-Lambert law? Is it followed for the data on the absorbance of
p-nitrophenol?
2. How would you determine from your data whether either the acid or alkaline
slope of your pH-activity curve is due to enzyme instability?
3. Predict the nature of pH-activity curves, based on fixed 15 min and 60 min
reaction times under the following conditions:
(a) enzyme is stable at all pH values
(b) enzyme slowly loses activity equally at all pH values
(c) enzyme loses activity more rapidly at higher than at lower pH values.
Hint: Plot product formed per min versus pH.
4. What other substrates might be used in this experiment?
8
EXPERIMENT 7
pH Optimum of an Enzyme
Laboratory Report
Name:
Date: _____________________
A. Standard curve
1.
Tube No.
Nitrophenol
concentration
(M )
Absorbance
1
2
3
4
5
6
7
0
0
Absorbance /
nitrophenol
concentration
(M -1)
0
2. Submit a graph in which absorbance readings (y-axis) are plotted versus pnitrophenol concentration (x-axis). Calculate the molar extinction coefficient for pnitrophenol. See Appendix C for discussion of proper graphing.
B. pH optimum
1. Submit a graph (or graphs) in which absorbance readings (y-axis) are plotted
versus time at each pH.
2. Determine initial rates from the above graph expressing them as moles/liter/min.
From this calculate percent hydrolysis of substrate after one min at each pH.
9
Tube No.
Measured pH
Initial rate
(M min-1)
Percent
hydrolysis at 1
min
1
2
3
4
5
6
7
8
3. Submit a graph of rate (M min-1) versus pH. Indicate what you consider to be
the pH optimum.
4. Submit answers to questions.
10