Hydrolysis of p-nitrophenyl acetate: estimation of rate enhancement by various catalysts.

Abstract: The rate enhancement of the hydrolysis of p-nitrophenyl acetate (PNPA) by seven catalysts, four amino acids and three enzymes, was determined at pH 7.0 and pH 8.0. All seven catalysts showed significant rate enhancement at both pH values. The enzymes, chymotrypsin, trypsin, and lysozyme, exhibited rate enhancement values greater by several orders of magnitude than those of the individual amino acids, serine, histidine, tyrosine, and lysine. Slight differences were noted in rate enhancement at the two different pH values, but the relative order of effectiveness among the seven catalysts was not changed.

Key Words: Enzymes, p-nitrophenyl acetate, chymotrypsin, trypsin, lysozyme, catalysis, rate enhancement, guanidine hydrochloride, denaturation 

Hydrolysis of p-nitrophenyl acetate (PNPA) to produce p-nitrophenol and acetic acid occurs readily.

p-nitrophenyl acetate + [H.sub.2]O [right arrow] p-nitrophenol + acetic acid

At neutral or slightly basic pH, p-nitrophenoxide ion (PNP) is produced, the absorbance of which can be measured in the visible region of the spectrum by simple spectrophotometers. The extent of hydrolysis of PNPA with time can thus be readily determined by measurement of PNP production.

PNPA has been used as a substrate in kinetic studies of several enzymes including bovine carbonic anhydrase II, [alpha]-chymotrypsin, and acetylcholine esterase (1). Head, et. al used PNPA in studies of the catalytic effectiveness of several enzymes and other catalysts at pH 7.0 (5). We have extended the work of Head, et. al by using additional enzymes and amino acids as catalysts as well as testing their relative effectiveness as catalysts not only at pH 7.0 as Head, et. al had done but also at pH 8.0.

Materials

Buffers were prepared using potassium phosphate, dibasic, I-3252, from the J. T. Baker Chemical Company, and potassium phosphate, monobasic, P-285, from Fisher Scientific Company. P-Nitrophenyl acetate, N-S 130, trypsin, T-82253, chymotrypsin, C-3 142, and lysozyme, L-6876, were purchased from Sigma, St. Louis, MO. Histidine (#17080) was purchased from the United States Biochemical Corporation and DL-serine (#5688) and Llysine monohydrochloride (#6467) were purchased from the Nutritional Biochemical Corporation. DL-Tyrosine (#5992) was purchased from Eastman. All reagents were used as received.

Stock solutions of PNPA were prepared to be 8.30 x [10.sup.-4] M and those of seine, tyrosine, lysine, and histidine were prepared to be 5.00 x [10.sup.-4] M. Stock solutions of trypsin, lysozyme, and chymotrypsin were prepared using 0.0050 g of the solid enzyme per 100 mL of solution. Phosphate buffers of pH 7.0 and 8.0 were prepared to be 0.0500 M.

Method

The procedure of Head, et. al was used to follow the hydrolysis of PNPA by measurement of the absorbance of the test solutions, over time, at 405 nm, using a Fisher Scientific Spectro Master, Model 415 spectrophotometer (5). Table 1 illustrates the preparation of the test solutions from the stock solutions. The absorbance of each solution was measured at 405 nm immediately after preparation and periodically over a 60-minute period at ambient temperature.

Results

Table 2 contains data collected for seven catalysts at pH 7.0 and Table 3 contains data collected for the same seven catalysts at pH 8.0. The use of a control solution containing no catalyst is quite necessary since PNPA readily undergoes hydrolysis in aqueous solution, as can be noted from the tabular data. The data reported by Head, et. al was indicated to be typical of their determinations (5). Similarly, we report one set of typical data obtained by the student involved in this project.

Figure 1 illustrates the progress of hydrolysis of PNPA, catalyzed by four different amino acids at pH 7.0 over the 60-minute test period. Figure 2 illustrates the progress of hydrolysis of PNPA, catalyzed by the same four amino acids, at pH 8.0.

Figure 3 illustrates the progress of PNPA hydrolysis, catalyzed by three different enzymes at pH 7.0. Figure 4 illustrates the progress of hydrolysis of PNPA, catalyzed by the same enzymes at pH 8.0. The graphs illustrated in Figures 1-4 were generated using Quattro Pro. The graphed data were not linearly transformed in order to show typical deviation of data points from linearity. Quattro Pro can, however, be used to fit the data to a least squares line.

Discussion

To better compare the effectiveness of the seven different catalysts used in the PNPA hydrolysis experiments, a rate enhancement (R.E.) value was calculated for each. The rate enhancement is the change in absorbance, with respect to the control, per minute, per mole of the catalyst in the assay solution. Units are [min.sup.-1][mol.sup.-1].

R. E. = [Rate.sub.catalyst] - [Rate.sub.control]/Moles of catalyst

The rate of reaction is reflected by the increase in absorbance of the test solutions per minute, which is due to the production of PNP as hydrolysis of PNPA occurs. Since change in absorbance per minute is the slope of each individual line on the graphs, the Quattro Pro program was used to obtain the slope of each line by means of regression analysis.

For example, 1.0 mL of the 5.00 x [10.sup.-4] M histidine stock solution was mixed with 2.0 mL of buffer and 1.0 mL of PNPA stock solution. The resulting test solution therefore contained 5.0 x [l0.sup.-7] mol of histidine. Regression analysis of the pH 7.0 data indicated a slope for the control of 0.0038896 [min.sup.-1] and a slope for the histidine data of 0.004294 [min.sup.-1]. The rate enhancement for histidine, then, is:

R. E. = (0.004294 -- 0.0038896)[min.sup.-1]/5.00 x [10.sup.-7] mol = 809 [min.sup.-1][mol.sup.-1]

The moles of enzyme in the assay solutions were calculated using molar masses of 21,600 for chymotrypsin, 14,300 for lysozyme, and 23,800 for trypsin (8, 2, 3). Different sources report somewhat different values for the molar masses of enzymes, but the rate enhancement values should not be drastically altered by slightly different molar masses.

Table 4 shows rate enhancement values calculated for the seven catalysts at pH 7.0, while Table 5 shows rate enhancement values for those same catalysts at pH 8.0.

From the data in Tables 1 and 2 and the graphs illustrated in Figures 1 and 2, it appears that all four amino acids exerted a catalytic effect in the hydrolysis of PNPA. Seine was most effective of the four amino acid catalysts and tyrosine was least effective. The relative order of effectiveness of these catalysts was the same at pH 8.0 as it was at pH 7.0. The rate enhancement values shown in Tables 4 and 5 indicate that tyrosine and histidine were less effective at pH 8.0 than at pH 7.0. Lysine and serine, however, appear to be more effective at the higher pH.

From the data in Tables 1 and 2 and the graphs illustrated in Figures 3 and 4, one may note that all three enzymes are better catalysts for the hydrolysis of PNPA than are any of the four amino acids used. Chymotrypsin and trypsin showed the greatest enhancement of catalytic activity. At pH 8.0, the catalytic activity was somewhat decreased compared to that at pH 7.0, but the relative order of catalytic effectiveness of the three enzymes was not altered. The decrease in activity at pH 8.0 may be due to alteration of the conformation of the enzymes such that the active site of the enzymes is less accessible by the PNPA substrate.

In an attempt to compare values of rate enhancement determined in this study to those of Head, et. al, it was noted that the values reported by Head, et. al must be multiplied by [10.sup.3] (2). This was determined by using their data and calculating rate enhancement in units of [min.sup.-1][mol.sup.-1]. Thus, their value of 1.70 [min.sup.-1][mol.sup.-1] for the rate enhancement of the amino acid serine is really 1.70 x [10.sup.3] [min.sup.-1][mol.sup.-1]. This compares well to the value of 1.682 x [10.sup.3] [min.sup.-1][mol.sup.-1]. determined for serine in this study. The value of 3.348 x [10.sup.3] [min.sup.-1][mol.sup.-1]. found by Head, et. al for the rate enhancement of chymotrypsin is really 3.348 x [10.sup.6] [min.sup.-1][mol.sup.-1]. and is the same order of magnitude as the value of 1.51 x 106 [min.sup.-1][mol.sup.-1]. determined in this study. There are several factors that may not allow exact duplication of the values reported by Head, et. al. Head and coworkers did not report the temperature at which their determinations were made so one must assume they were made at room temperature. It is possible that temperatures were somewhat different in their Manchester laboratory than those in our laboratory. Additionally, it is uncertain that the purity of the catalysts used in both studies were identical. As indicated elsewhere in this paper, some differences may exist in the molar masses used for various enzymes in calculations of rate enhancement values.

Trypsin and chymotrypsin are serine proteases that have been shown to follow a Ping Pong Bi Bi catalytic mechanism (4, 6, 7). During the catalysis of the hydrolysis of PNPA, an active site serine residue of chymotrypsin is acylated, with the release of PNP, followed by release of the acetate group from the acyl-enzyme intermediate due to the action of the second substrate, water. The latter step is rate-determining.

[FORMULA NOT REPRODUCIBLE IN ASCII]

One might speculate that lower [[H.sup.+]], that is, higher pH, might serve to help drive these steps of the mechanism, but that is not reflected by the decreased hydrolysis noted at pH 8.0 compared to that at pH 7.0. Alteration of the enzyme to a slightly less active conformation may be a more likely cause.

Although lysozyme shows noticeable catalytic activity in the hydrolysis of PNPA, it is the least effective of the three enzymes tested. It is considerably more effective than any of the four amino acids tested, however. While lysozyme is a hydrolase, its normal substrate is neither an ester nor a protein, but the glycosidic linkages of N-acetylmuramic acid (NAM) that is linked to N-acetylglucosamine (NAG) in bacterial cell walls. (8) Serine residues are not thought to be involved in the active site binding and catalysis by lysozyme.

Conclusion

Anderson, et. al, and Head, et. al have indicated that the study of the catalytic hydrolysis of p-nitrophenyl acetate has been the basis for a number of projects by students. (1, 5) We agree that these kinds of studies can make interesting projects for students and have used them as such for the past two years. We have extended the studies of Head, et. al by using different amino acids as well as other hydrolytic enzymes at two different pH values, 7.0 and 8.0. While all seven catalysts showed significant rate enhancement, the enzymes were the most effective. Of the enzymes, the seine proteases were the most effective in catalyzing hydrolysis of PNPA. Of the four amino acids used as catalysts, serine was the most effective at both pH values.

The most significant feature of the procedure is that it allows direct comparison of the catalytic effectiveness of potential catalysts. Comparisons must be made under the same set of conditions, with a control used with each set of test solutions. It seems possible that such studies could also be adapted and used as a specific experiment in the biochemistry laboratory rather than as a full semester project. Preparation of test solutions prior to the laboratory period would allow laboratory time to be used for data collection. Data analysis and preparation of reports could be done out of class.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Table 1

Composition of Test Solutions

Solution       Catalyst     PNPA     Buffer,  Total Volume,
Type           Stock, mL  Stock, mL    mL          mL

Control          None        1.0       3.0         4.0
Test Solution     1.0        1.0       2.0         4.0

Table 2

Absorbance of Test Solutions @ 405 nm at pH 7.0

Time,           0       5       10      15      20      25      35
min

Control       0.0482  0.0506  0.0555  0.0580  0.1024  0.1249  0.1838
Tyrosine      0.0506  0.0605  0.0680  0.1079  0.1135  0.1367  0.2076
Histidine     0.0531  0.0655  0.0731  0.1192  0.1279  0.1805  0.2111
Lysine        0.0555  0.0706  0.0996  0.1296  0.1561  0.1904  0.2549
Serine        0.0706  0.0809  0.1163  0.1612  0.1858  0.2218  0.2757
Chymotrypsin  0.2007  0.2255  0.2924  0.3372  0.3615  0.4437  0.4881
Lysozyme      0.1612  0.1772  0.2403  0.2557  0.3054  0.3468  0.4202
Trypsin       0.2111  0.2403  0.3188  0.3872  0.4034  0.4815  0.5302

Time,           45      60
min

Control       0.2147  0.2518
Tyrosine      0.2366  0.2716
Histidine     0.2480  0.2924
Lysine        0.2840  0.3098
Serine        0.3054  0.3279
Chymotrypsin  0.5686  0.6198
Lysozyme      0.4815  0.5376
Trypsin       0.5935  0.6576

Table 3

Absorbance of Test Solutions @ 405 nm at pH 8.0

Time,           0       5       10      15      20      25      35
Min

Control       0.0410  0.0506  0.0555  0.0731  0.1024  0.1163  0.1427
Tyrosine      0.0842  0.0555  0.0580  0.0783  0.1079  0.1278  0.1457
Histidine     0.0506  0.0605  0.0703  0.0915  0.1107  0.1367  0.1612
Lysine        0.0531  0.0610  0.0742  0.0996  0.1249  0.1487  0.1925
Serine        0.0630  0.0706  0.0862  0.1135  0.1427  0.1707  0.2147
Chymotrypsin  0.1192  0.1278  0.1397  0.1612  0.1772  0.2518  0.3098
Lysozyme      0.0996  0.1079  0.1135  0.1278  0.1487  0.1643  0.2366
Trypsin       0.1308  0.1367  0.1487  0.1739  0.2007  0.2676  0.3372

Time,           45      60
Min

Control       0.1805  0.2007
Tyrosine      0.1871  0.2291
Histidine     0.1904  0.2366
Lysine        0.2366  0.2557
Serine        0.2480  0.2924
Chymotrypsin  0.3420  0.4318
Lysozyme      0.3143  0.4089
Trypsin       0.3872  0.4498

Table 4

Rate Enhancement Values for Various Catalysts at pH 7.0

              Regression           Moles of Catalyst
Catalyst      Slope, [min.sup.-1]        in the
                                     Test Solution

Control       0.0038896
Tyrosine      0.004067716          5.00 x [10.sup.-7]
Histidine     0.004294             5.00 x [10.sup.-7]
Lysine        0.0046699            5.00 x [10.sup.-7]
Serine        0.004730683          5.00 x [10.sup.-7]
Chymotrypsin  0.007376             2.31 x [10.sup.-9]
Lysozyme      0.006843             3.50 x [10.sup.-9]
Trypsin       0.007667             2.10 x [10.sup.-9]

                 Rate Enhancement,
Catalyst      [min.sup.-1][mol.sup.-1]


Control
Tyrosine         356
Histidine        809
Lysine           1561
Serine           1682
Chymotrypsin     1.51 x [10.sup.6]
Lysozyme         0.844 x [10.sup.6]
Trypsin          1.80 x [10.sup.6]

Table 5

Rate Enhancement Values for Various Catalysts at pH 8.0

                  Regression       Moles of Catalyst
Catalyst      Slope, [min.sup.-1]        in the
                                     Test Solution

Control            0.00292
Tyrosine           0.002915        5.00 x [10.sup.-7]
Histidine          0.003216        5.00 x [10.sup.-7]
Lysine             0.003791        5.00 x [10.sup.-7]
Serine             0.004134        5.00 x [10.sup.-7]
Chymotrypsin       0.005572        2.31 x [10.sup.-9]
Lysozyme           0.005343        3.50 x [10.sup.-9]
Trypsin            0.005945        2.10 x [10.sup.-9]

                 Rate Enhancement,
Catalyst      [min.sup.-1][mol.sup.-1]


Control
Tyrosine                -10
Histidine               592
Lysine                  1742
Serine                  2428
Chymotrypsin     1.15 x [10.sup.6]
Lysozyme         0.692 x [10.sup.6]
Trypsin          1.44 X [10.sup.6]

Allen Scism (1)

(1.) Corresponding author.

Literature Cited

(1.) Anderson, J., Byrne, T., Woelfel, K. J., Meany, J. E., Spyridis, G. T., and Pocker, Y., J. Chem. Educ. 1994, 71, 715-718.

(2.) Head, Michael B., Mistry, Kalpna S., Ridings, Bernard J., and Smith, Christopher A., J. Chem. Educ. 1995, 72, 184-186.

(3.) Horton, Robert H., Moran, Laurence A., Ochs, Raymond S., Rawn, I. David, and Scrimgeour, K. Gray, Principles of Biochemistry, Neil Patterson Publishers/Prentice-Hall, Inc. Englewood Cliffs, NJ, 1993, p 5.16.

(4.) Hartley, B. S., and Kilby, B. A., Biochem. 1. 1954, 56, 294.

(5.) Voet, Donald, and Voet, Judith G., Biochemistry, John Wiley & Sons, New York, 1990, pp. 373-375.

(6.) Voet, Donald, Voet, Judith G., and Pratt, Charlotte W., Fundamentals of Biochemistry, John Wiley & Sons, Inc., New York, 1999, pp. 300-307.

(7.) Biochemicals and Reagents for Life Science Research, Sigma-Aldrich Co., St. Louis, MO, 2000, p.1988.

(8.) Dixon, Malcolm, and Webb, Edwin C., Enzymes, Academic Press, Inc., New York, 1964, p. 453.