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Department of Biochemistry, Oklahoma State University, Stillwater, Oklahoma 74078
and
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66502
Chloroperoxidase and lactoperoxidase actively halogenate tyrosine residues of protein substrates and cause inactivation of a-amylases from B. subtilis, barley, hog pancreas and Aspergillus oryzae. Chlorotyrosine is produced in the inactivation of barley a-amylase. The B. subtilis enzyme is rapidly inactivated by chloroperoxidase in a process requiring either hydrogen peroxide, chloride and enzyme or sodium chlorite and enzyme. About 1.3 moles of peroxide is required per g-mole (48 × 103 g) for 95% inactivation of the dimeric enzyme. No chloride incorporation was found during inactivation of B. subtilis a-amylase. Amino acid analyses of B. subtilis a-amylase after acid and basic hydrolysis indicated methionine to be the only amino acid significantly modified as a result of the inactivation.
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Chemical modification is an important tool for studying structure-function relationships of enzymes and other biologically active proteins (1). The major problem encountered with this technique is the lack of specificity or selectivity of the chemical reagents. Since most reagents show reactivity toward more than one type of amino acid, the use of enzymes as modifying agents should bring about enhanced specificity in most cases. These specificities should be increased by steric limitations involved in bringing large proteins into close proximity for reaction. Therefore, enzyme modification techniques should offer significant advantages for studying enzyme functional groups.
Using chemical modification Connellan and Shaw (2) have implicated a tyrosine residue as being critical in B. subtilis a-amylase. This raises the possibility that a tyrosine residue may function as a critical amino acid in amylases. In order to investigate this assumption, two different approaches have been used:
1) A series of a-amylases have been subjected to treatments with lactoperoxidase and chloroperoxidase.
2) The inactivation of B. subtilis a-amylase by chloroperoxidase has been investigated in more detail. Both lactoperoxidase and chloroperoxidase inactivate all of the a-amylases studied, a result which is consistent with the occurence of critical tyrosine. However, the chloroperoxidase inactivation of B. subtilis a-amylase may not involve tyrosine. These results show the utility of enzymatic modification procedures with the peroxidases, but they also indicate the caution which must be used in interpreting results in terms of specific inactivation mechanisms.
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Lactoperoxidase from bovine milk was purchased from Sigma Chemicals (St. Louis), and chloroperoxidase was isolated from Caldariomyces fumago (3). B. subtilis type II-A, hog pancreatic and Aspergillus oryzae type IV-A a-amylases were purchased from Sigma Chemical. Barley a-amylase was prepared by the procedure of Mitchell (4).
Analytical Procedures
a-Amylase activity was measured using a modification of the procedure of Schuster and Gifford (5). One ml of 10 mM sodium acetate buffer, pH 4.8, with 0.01 M CaCl2 is added to 1 ml of 1.4 mg/ml Merck solu-
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ble starch (special for diastatic determination) dissolved in the same buffer. A 10-µl quantity of enzyme solution is added, followed by appropriate buffer to make a 3-ml volume; the mixture is then incubated at 30 C for 1 min. and quenched with 1 ml 2.5 mM KI with 5 mM I2 solution in 50 mM HCl. Before reading the absorbance at 620 nm, the assay solution is diluted with 5 ml H2O. Amylase activity was also measured by the "Azure Amylose" procedure (6).
Protein concentrations were determined by the method of Lowry et al. (7). The activity of chloroperoxidase was measured using the activity assay of Morris and Hager (3) and was about 23.8 × 104 units/ml (1 unit = 1 µ mole dichlorodimedone formed/ min. at room temperature). Protein samples for amino acid analysis were hydrolyzed in 6 N HCl at 110 C for 24 hr. (8) or in 4 N NaOH at 100 C for 8 hr. (9). Analyses were performed on a Beckman 120 C amino acid analyzer.
Barley (4) and B. subtilis (10) a-amylasewere judged to be homogeneous by gel electrophoresis and ultracentrifugation.
Lactoperoxidase Treatment of a-Amylases from B. subtilis, Aspergillus oryzae, Hog Pancreas and Barley
A 0.1-ml quantity of a-amylasesolution (10.0 mg/ml) in 10 mM acetate buffer, pH 4.8, with 10 mM CaCl2 was incubated with 0.2 ml of 5 mM KI, 10 µl of 0.01 mM H2O2, 5 µl lactoperoxidase solution (1 mg/ml) and 0.7 ml 50 mM acetate buffer, pH 5.0, at 37 C. Three blanks were used, one without lactoperoxidase, one without KI, and one without a-amylase. At 10-min. intervals 10-µl aliquots were removed for activity assay.
Chloroperoxidase Treatment of a-Amylase
The reaction mixture containing 1.0 mg of amylase, 10 mM in Ca (NO3)2, 60 mM in NaCl and 2 mM in H2O2 in 2.0 ml of 10 mM acetate buffer (pH 4.8) was incubated at 37 C with 5 µl of chloroperoxidase (1.2 mg/ml, 1220 units). Three blanks were used, one without chloroperoxidase, one without NaCl, and one without a-amylase. After 10-min incubation, 10-µl aliquots were removed for amylase activity assay.
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Inactivation of Amylase with Lactoperoxidase and Chloroperoxidase
All the a-amylases investigated can be inactivated by either lactoperoxidase or chloroperoxidase (Table 1). The reactions require enzyme, peroxidase and halide. Deletion of any of these from the reaction mixture decreased or eliminated the inactivations. Some inactivation of the pancreatic and A. oryzae amylases is observed in the absence of the modifier enzymes, suggest-
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ing an oxidative effect of peroxide alone. Neither the barley nor B. subtilis enzymes are inactivated under these conditions. Both enzymes show homogeneity as determined by gel electrophoresis and ultracentrifugation (4, 10). It is questionable whether these effects involve direct action on the enzymes, since the inactivated enzymes are less highly purified preparations than the other two and may contain other oxidizable substances. After 10 min, maximum inactivation had occurred; therefore, all peroxidase reactions were conducted with 10-min incubation. The B. subtilis inactivation with chloroperoxidase was essentially completed after 1 min.
The modification of tyrosine does occur under the conditions used, as shown by
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amino acid analysis for chlorotyrosine of the barley a-amylase or bovine serum albumin (Table 2). Complete amino acid analysis of barley a-amylase that had been treated with chloroperoxidase showed no significant change in amino acid residues other than tyrosine (Table 3).
Inactivation of B. subtilis a-Amylase
Because the amylase from B. subtilis can be obtained in highly purified form in quantity, a more thorough examination of the inactivation of this enzyme was undertaken (see Figure 1). In the presence of chloroperoxidase and excess chloride, 1.0 mg of a-amylase is maximally inhibited by 0.05 µl of 0.5 mM H2O2. Thus the amount of peroxide required for 75% inactivation is slightly greater than one mole of peroxide per mole of enzyme protein, on the basis of a subunit molecular weight of 24 × 103 (10, 11). Figure 2 shows the inactivation of B. subtilis a-amylase to depend on the presence of chloride and chloroperoxidase.
Analysis of B. subtilis a-amylasefor chlorotyrosine by amino acid analysis was negative (Table 2). Likewise, less than 0.1 mole of 36Cl was incorporated per mole of B. subtilis amylase that was 93% inactivated, as measured by the starch-iodine amylase assay. These results suggest that the peroxidase is inhibiting amylase by oxidation rather than chlorination. Inactivated amylase was therefore analyzed for histidine and tyrosine by amino acid analysis and for tryptophan and tyrosine by the method of Edelhoch (12). As shown in Table 3 no loss of these amino acids was found in the inactivated enzyme. We also treated chloroperoxidase with N-acetyltryptophan, N-acetyltyrosine and N-acetylhistidine and observed no modification. This is consistent with the observation that there is no incorporation of radioactively labeled chloride and that spectrophotometric determination of tyrosine and tryptophan shows no differences between the chloroperoxidase-treated and untreated B. subtilis a-amylase.
Sulfur-containing amino acids are also susceptible to oxidative processes (16). B. subtilis a-amylase contains no free sulfhydryl or disulfide groups (13), but it does have methionine. Since methionine sulfoxide, the product of limited oxidation of this amino acid, may be converted back to methionine by acid hydrolysis, amino acids were analyzed after basis hydrolysis of different samples of native and inactivated enzymes. The native enzyme consistently showed 4-5 methionines per mole, while the inactivated enzyme showed only 2-3 residues/mole of protein (Table 4). These results indicate that 1-2 methionines
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are altered in the inactivated enzyme. This is the result of 8 independent amino acid analyses of separate experiments.
Another interesting facet of the inactivation is the chloride requirement (Table 5). This suggests that the peroxidase intermediate in the inactivation reaction is an oxidized form of chlorine rather than oxidized enzyme. Examination of the effects of various oxidized-chlorine salts shows that only NaClO2 will substitute for H2O2 in the enzyme inactivation. This effect is consistent with studies showing that an oxygenated form of chloride is the active intermediate in the catalytic mechanism (7).
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B. subtilis a-amylase is rapidly and specifically inactivated by treatment with chloroperoxidase in a process requiring either enzyme plus chloride and peroxide or enzyme plus chlorite. This inactivation does not occur with the production of chlorotyrosine as in the case of barley a-amylase, which has 24% of its tyrosine residues converted to chlorotyrosine. Since B. subtilis a-amylase tyrosine residues are not modified by chloroperoxidase and also since 36Cl is not incorporated into the protein, then perhaps either tyrosine is oxidized or another residue necessary for catalysis or binding is inactivated. Our data suggest that another amino acid residue in B. subtilis a-amylase which may be involved catalytically is modified by chloroperoxidase.
Two factors suggest that the inactivation does not occur by direct action of the enzyme on a key catalytic residue. First, maximal inactivation requires only slightly more than stoichiometric quantities of peroxide per mole of enzyme, a molecular weight of 48 × 103 being assumed (10). Second, the sigmoidal nature of the inactivation curve indicates that most of the stoichiometric amount of peroxide can be utilized with virtually no inactivation of the enzyme. An alternative explanation of these results is that modification occurs at one or more residues of one or more of the protomers of the dimeric enzyme to cause a conformation change resulting ultimately in almost total loss of the enzyme activity. This explanation would account for the limited peroxide requirement and the sigmoidal nature of inactivation. Another interesting observation is that chloroper-
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oxidase inactivation is not reversed in the presence of substrate. That is, there is no protection by substrate. This also suggests that the inactivation occurs at noncatalytic residues. Methionine appears to be the amino acid modified, although we did not obtain a direct correlation of methionine oxidation and enzyme inactivation other than by amino acid analysis for methionine residues. We did not observe stoichiometric amount of methionine sulfoxide; however, amino acid analysis of chloroperoxidase-treated B. subtilis a-amylase showed a decrease in the number of methionine residues per 103 amino acid residues. Sulfur-containing amino acids interfere with the peroxide-catalyzed halogenation, iodide oxidation, and tyrosine halogenation (16). In the case of methionine the mechanism may involve a methionyl-sulfonium derivative.
Also notable is the relative stability of barley a-amylase enzymes. An even more interesting observation is the comparison of A. oryzae and B. subtilis a-amylase; both contain the same relative amount of methionine and are equally susceptible to chloroperoxidase, but they respond differently to lactoperoxidase. We might also add that the A. oryzae a-amylase is quite different from B. subtilis a-amylase inasmuch as A. oryzae enzyme contains sulfhydryl groups and a carbohydrate moiety (15) whereas B. subtilis a-amylase has neither cysteine (13) residues nor a carbohydrate moiety.
Although these peroxidases are often used for halogenation reactions, the inactivation of B. subtilis a-amylase apparently involves an oxidation process with no halogenation. On the other hand, the inactivation of barley a-amylase involves halogenation of tyrosine. The oxidative capabilities of these halogenating enzymes must be considered in any reaction system involving a functional activity. Unfortunately, oxidative reactions are much more difficult to observe because incorporation of radioactive substrates cannot be used to follow the reaction. Nevertheless, this study does indicate the usefulness of the peroxidative enzymes in uncovering useful and interesting information concerning structure-function relationships of other enzymes. In particular this is the first recognition of specific amino acid residues involved with the catalytic properties of these carbohydrate-hydrolyzing enzymes. At this moment the mechanism of catalysis is still elusive.
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1. G. E. MEANS and R. E. FEENY, Chemical Modification of Proteins, Holden Day, San Francisco, 1971.
2. J. M. CONNELLAN and D. C. SHAW, J. Biol. Chem. 245: 2845-2851 (1970).
3. D. R. MORRIS and L. R. HAGER, J. Biol. Chem. 241: 1763-1768 (1966).
4. E. D. MITCHELL, Phytochemistry 11: 1673-1676 (1972).
5. L. SCHUSTER and R. H. GIFFORD, Arch. Biochem. Biophys. 96: 534-540 (1962).
6. H. RINKERKNECHT, P. WILDING, and B. J. HAVERBACK, Experientia 23: 805 (1967).
7. O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDELL, J. Biol. Chem. 193: 265-275 (1951).
8. S. MOORE and W. H. STEIN, Meth. Enzymol. VI: 819-831 (1963).
9. M. FLORKIN and E. H. STOTZ eds., Comprehensive Biochemistry 7: 65 (1963).
10. E. D. MITCHELL, P. RIQUETTI, R. H. LORING, and K. L. CARRAWAY, Biochem. Biophys. Acta 295: 314-322 (1973).
11. J. F. ROBYT and R. J. ACKERMAN, Arch. Biochem. Biophys. 155: 445-451 (1973).
12. H. EDELHOCH, Biochemistry 6: 1948-1954 (1967).
13. J. M. JUNGE, E. A. STEIN, H. NEURATH, and E. H. FISCHER, J. Biol. Chem. 234: 556-561 (1959).
14. P. F. HOLLENBERG, T. RAND-MEIR, and L. P. HAGER, J. Biol. Chem. 249: 5816-5825 (1974).
15. B. SEON, H. TODA, and K. NARITA, J. Biochem. 58: 348-354 (1965).
16. M. MORRISON and G. R. SCHONBAUM, Ann. Rev. Biochem. 45: 861-888.
17. W. D. HENESON and L. P. HAGER, J. Biol. Chem. 254: 3175-3181 (1979).
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