In the Laboratory
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Chemical Modification of Papain and Subtilisin: An Active Site Comparison An Undergraduate Biochemistry Experiment Mireille St-Vincent and Michael Dickman* Collège Universitaire de Saint-Boniface, Winnipeg, MB, Canada, R2H 0H7; *
[email protected] Enzyme chemistry is an integral part of any introductory biochemistry course and is an important aspect of most undergraduate protein chemistry courses. By studying the action of enzymes, students learn to appreciate the dynamic nature of proteins as well as their role catalyzing myriad chemical reactions in biological systems. Much time in class, especially at an introductory level, is spent investigating the catalytic cycle and the role of the various amino acid residues in the active site of the enzyme. While there are numerous enzyme systems that can be studied, proteases are often used in class as well as in teaching laboratories because they are relatively simple, inexpensive, and well understood. In spite of this, very few undergraduate experiments exist that directly explore the chemistry of the amino acid residues in an enzyme’s active site. (These readers found none.) Many biochemical techniques such as affinity labeling, radioactive tagging, and fluorescent and spin labeling take advantage of the chemical specificity of residue side chains to probe protein chemistry phenomena (1). Affinity labels are used to identify active site residues important in catalysis, and spin labeling is employed as a general method for exploring the conformation of membrane and soluble proteins (2). The reactive side chains of aspartate, glutamate, lysine, serine, threonine, tyrosine, histidine, and cysteine can be targeted with a wide variety of reagents. The experiment we are proposing uses methyl methanethiosulfonate (MMTS) and phenylmethylsulfonyl fluoride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilisin, respectively (3–5). Both enzymes are subjected to the same series of treatments, and the differences are noted. The chemical modification of papain by MMTS causes a complete loss of activity, and when the active site cysteine is regenerated, the return of activity is noted. Since subtilisin is a serine protease, treatment with MMTS has no effect on it. However, treatment of subtilisin with PMSF results in an irreversible loss of activity while the sulfonyl fluoride does not dramatically affect papain under the reaction conditions used in this experiment. The comparison of papain and subtilisin accomplishes many desirable teaching goals. The active site differences between the two proteases are displayed. This opens many possibilities for discussion regarding the reactivity of cysteine versus serine and the role of cysteine in proteins. (Subtilisin contains no cysteine while papain has seven.) The covalent modification of the enzymes and subsequent rescue of papain shows the beginning biochemist that proteins can be manipulated structurally and that this directly affects their functioning. In addition, students will appreciate the ease of manipulating the two enzymes as if they were simple chemi1048
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cal reagents. As an added plus, substrate specificity can be discussed, since the enzyme specificities are different, and the characteristics of the two inhibitors can be compared as well. All this is possible with a few relatively inexpensive reagents and a spectrophotometer. Experimental Procedure Papain and subtilisin were obtained as crystalline powders from Sigma. MMTS, PMSF, cysteine, and the two substrates, Nα-benzoyl-DL-arginine p-nitroanilide hydrochloride and N-succinyl-L-phenylalanine p-nitroanilide, were also Sigma products. Stock solutions of the enzymes were prepared from acetate buffer (pH 5.0, 50 mM). Calcium ion was added to subtilisin by the addition of CaCl2 (10 mM) to the stock solution as well as immediately after dialysis. Enzyme assays were conducted in sodium phosphate buffer (pH 7.6, 50 mM) and activity was monitored by measuring the formation of p-nitroaniline at 410 nm. Stock solutions of the substrates were prepared in dimethyl sulfoxide (DMSO), and the final concentration of DMSO in the assays was 1%. Subtilisin was assayed using N-succinyl-L-phenylalanine p-
cys-25 side chain COO S S
papain
−
+
and
NH3
papain
about 90%
SH
about 10%
excess cysteine
COO papain
SH
+
H3N
+
S S COO
almost 100%
+
NH
−
cystine
Scheme I. Liberation of papain’s active site with excess cysteine.
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In the Laboratory
nitroanilide while papain was assayed using Nα-benzoyl-DLarginine p-nitroanilide hydrochloride. MMTS was diluted in acetonitrile (CH3CN), and PMSF was used as a stock solution in ethanol. All modification reactions were conducted in acetate buffer at pH 5.0. Dialysis was done in 5 mM acetate buffer at pH 5.0 with 50 mM NaCl using a molecular weight cutoff of 6,000 to 8,000. All enzyme solutions and modification reactions were prepared and conducted in polypropylene tubes fitted with snap-on polyethylene caps. All enzyme assays were done in disposable polystyrene cuvettes. The substrate stock solutions as well as the dilution of MMTS and PMSF were made in siliconized micro centrifuge tubes. A detailed experimental procedure is available in the Supplemental Material.W Hazards Protein powders are allergenic so students were given polypropylene tubes with 20 mg of enzyme. They could then add the acetate buffer directly to the tube at the beginning of the experiment. CH3CN can irritate eyes, nose, throat, and skin. DMSO is absorbed directly through the skin and acts as a solvent carrier for the transport of any substance in solution. MMTS has a strong odor and is irritating to the eyes, skin, and respiratory system. In addition, PMSF burns the skin and eyes, and is very destructive to mucous membranes. Gloves, labcoat, and eye protection should be worn when manipulating these four chemicals. The solution of MMTS in CH3CN should be manipulated in a fume hood. Results and Discussion Commercial papain often contains a significant quantity of the mixed disulfide of papain and cysteine (6). As a
result, the initial activity of papain can be as low as 10% of maximum activity (7). For this reason, the enzyme solutions are first treated with excess cysteine to fully liberate papain’s active site cysteine-25. Though this step is not necessary for the success of the experiment, the activity of papain will seem to increase 10-fold after chemical modification and subsequent regeneration of the active site cysteine, if it is omitted. This may confuse students, so it was deemed prudent to first obtain 100% active papain before modifying with MMTS (Scheme I). Also, the change from 100% activity to zero activity and back to 100% activity gives a much more dramatic effect than 10% to zero to 100% activity. If this initial treatment with cysteine is done by the instructor, then the experiment can be easily performed in a three-hour period. After liberating cysteine-25, the enzymes are assayed with their respective substrates. Immediately following this step, both enzyme solutions are split in two samples. MMTS and PMSF are added to a sample of each enzyme solution (Scheme II). While a pH of 5.0 appears low for the reaction between papain and MMTS, it has been shown that cysteine25 of papain and MMTS react at a relatively constant rate between pH 4.5 and 7.5 (8). PMSF can be used at low pH to titrate the active sites of serine proteases (9). They are given half an hour to react before freezing them in either liquid nitrogen or a dry ice and ethanol bath. (It is important to quick-freeze protein solutions to avoid enzyme denaturation.) The frozen enzymes are kept at ᎑20 ⬚C until the second period. After thawing the enzymes at the beginning of the second period, dialysis is done to eliminate the excess MMTS, PMSF breakdown products as well as any other small molecular weight compounds. Care must be taken during dialysis and opening of the dialysis tubes to avoid introducing extraneous water into the enzyme solutions, which would
cys-25 side chain
papain
ser-221 side chain
SH
MMTS
MMTS
PMSF
O S papain
S
CH3
O subtilisin
O ⬃100 % yield
unchanged
and
O S
OH
subtilisin
S
⬃100 %
PMSF
O S
papain
OH
subtilisin
unmodified papain
O MMTS ⬅ H3C
O
S
S
CH3
PMSF ⬅
CH2
O
S
F
O
Scheme II. Treatment of papain and subtilisin with MMTS and PMSF.
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In the Laboratory Table 1. Student Activity Resultsa for the Reaction between the Substrate and the Enzyme as Measured by the Absorbance of p-Nitroaniline Subtilisin and N-Succinyl-L-Phe p-nitroanilide Abs
Papain and Nα-Benzoyl-DL-Arg p-nitroanilide hydrochloride Abs
Commercial enzyme
0.382
0.294
After the first treatment with Cys
0.354
2.134
Enzyme Condition
MMTS
PMSFb
MMTS
PMSFb
After modification with MMTS or PMSF
0.312
0.007
0.016
1.35
After the second treatment with Cys
0.294
0.006
2.09
2.77
a
The activity is measured by the magnitude of the p-nitroaniline absorbance at 410 nm. Data are the average from five student‘s results.
b
The comparison with PMSF has not yet been attempted with students. The authors obtained these results.
lower their apparent activity. With the modified enzymes in hand, another round of assays is performed. This test should show zero or close to zero activity for papain with MMTS and for subtilisin with PMSF. No significant change in activity occurs for subtilisin with MMTS, and under the reaction conditions used in this experiment, papain is not efficiently inhibited by PMSF. At this point, the modified enzymes are treated with an excess of cysteine thus reducing the mixed disulfide, papain⫺S⫺SCH3 as well as any PMSF inhibition of papain. Neither subtilisin sample is affected by this treatment. A final assay is performed and the results are presented in Table 1. Results do vary for two reasons. It is very difficult to avoid adding water to the enzyme solutions when the dialysis tubes are opened. The additional water dilutes the enzymes, and causes a drop in their apparent activity. This effect is most easily seen for subtilisin. Also, only one assay is done at each step owing to the expense of the enzymes and substrates. Conclusion The experiment demonstrates how enzymes can be easily modified in a very specific manner. Commercial papain is brought to near 100% activity, then covalently modified with MMTS to render it completely inactive and finally reactivated. PMSF has limited effect on papain under the reaction conditions used in this experiment. Subtilisin is subjected to an identical series of treatments, but the MMTS
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has no effect while PMSF completely inhibits this enzyme irreversibly. The results serve to underline the different active site structures and mechanisms of the two enzymes. This is accomplished using inexpensive reagents and simple laboratory techniques. W
Supplemental Material
Detailed instructions for students and notes for instructors are available in this issue of JCE Online. Literature Cited 1. Fersht, A. Structure and Mechanism in Protein Science; Freeman: New York, 1999; Chapter 9. 2. Columbus, L.; Hubbell, W. L. TiBS, 2002, 27, 288–295. 3. Singh, R.; Blättler, W. A.; Collinson, A. R. Methods Enzymol. 1995, 251, 229–236. 4. Gold, A. M.; Fahrney, D. Biochemistry 1964, 3, 783–791. 5. Whitaker, J. R.; Perez-Villasenor, J. Arch. Biochem. Biophys. 1968, 124, 70–78. 6. Klein, I. B.; Kirsch, J. F. Biochem. Biophys. Res. Commun. 1969, 34, 575–581. 7. Singh, R.; Blättler, W. A.; Collinson, A. R. Anal. Biochem. 1993, 213, 49–56. 8. Roberts, D. D.; Lewis, S. D.; Ballou, D. P.; Olson, S. T.; Shafer, J. A. Biochemistry 1986, 25, 5595–5601. 9. Hsia, C. Y.; Ganshaw, G.; Paech, C.; Murray, C. J. Anal. Biochem. 1996, 242, 221–227.
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