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Maltosylamine, a specific inhibitor of β-amylase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 195, No. 2, July, pp. 392-395, 1979
Maltosylamine, DANIEL Department
a Specific
E. WALKER*
of Biochemistry,
Purdue
Inhibitor1 BERNARD
AND
University,
Received January
of /3-Amylase AXELROD3
West Lafayette,
Indiana
47901
22, 1979
P-Maltosylamine has been synthesized for the first time. It is an effective specific inhibitor of sweet potato /3-amylase. This result extends the observation that I-aminoglycosides are specific inhibitors of exoglycosidases which hydrolyze the corresponding glycose and also demonstrates that an enzyme acting with inversion, as well as those acting with retention of anomeric configuration, can be inhibited by glycosylamines. Maltosylamine, which acts as a reversible inhibitor of P-amylase, appears to be directed to the active site since it protects the essential sulfhydryl group of the enzyme from inactivation by N-ethylmaleimide.
P-amylase acts with inversion (5). We report herein the first recorded synthesis of maltosylamine and a study of its effects on P-amylase.
It has been previously demonstrated that 1-aminoglycosides competitively inhibit the corresponding glycosidase (1). Thus, glycosylamine inhibits glucosidase but not galactosidase, while galactosylamine inhibits galactosidase but not glucosidase. Until the present work, the specific inhibition of glycosidases by their corresponding glycosylamines has been observed only with those enzymes which release monosaccharides. Since it has been rigorously confirmed that P-amylase catalyzes the hydrolytic release of a disaccharide, p-maltose (2), from its natural substrate, cu-1,4-glucan, it was of interest to see if the 1-aminoglycoside of p-maltose would specifically inhibit this enzyme. It was already known that glucosylamine does not significantly inhibit this enzyme (3). It was also of special interest to examine the inhibition of P-amylase by maltosylamine because all previous observations of inhibition by glycosylamines have been made with glycosidases which release their glycosyl product with retention of anomeric configuration (1, 3, 4). It is well known that
MATERIALS
Materials. /%Amylase (sweet potato) and amylo-cu1,4-o-1,6-glucosidase (Aspergillus niger) were from Boehringer-Mannheim GmbH. a-Amylase (hog pancreas) was obtained from P. L. Biochemicals. Calbiochem supplied a-amylase (dextrinizing) from Bacillus subtilis. a-Methylglucosidase from Saccharomyces oviformis was purified by the method of Fukui and Axelrod (6). Maltase from the same organism adapted to maltose was used as a crude extract. N-Ethylmaleimide (NEM)” was obtained from Aldrich Chemical Company. Soluble starch (Lintner’s) was purchased from Fisher Scientific Company. Maltosylamine was Synthesis of maltosylamine. synthesized essentially by the general method which Isbell and Frush (7) have described for the preparation of monoglycosylamines. Ammonium chloride (1.0 g) was dissolved in 50 ml of anhydrous methanol. Maltose (80 g) was added and NH3 was bubbled through the mixture at 0°C until the sugar went into solution. The flask was stoppered and kept in the refrigerator at 4°C for 3 months during which time crystals formed. The product was recrystallized by dissolving in 2 parts HZ0 (40”) and adding 4 parts methanol and 2 parts isopropanol. The crystals which formed at 4” were collected by vacuum filtration and washed with ice-cold methanol saturated with NH, and were dried for 24 h irk oacuo over NaOH. In subsequent preparations, crystallization
* Supported by NIH Research Grant GM 22189. This is Journal Paper No. 7421, Purdue Agricultural Experiment Station. 2 Recipient of U. S. Public Health Predoctoral Trainee Fellowship GM 7211. Present address: Diagnostic Quality Control, Dow Chemical Company, P.O. Box 68511, Indianapolis, Indiana 46268. 3 To who requests for reprints may be sent. 0003-9861/79/080392-04$02.00/O
Copyright 0 1979 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
AND METHODS
4 Abbreviation 392
used: NEM, N-Ethyl
maleimide.
MALTOSYLAMINE.
A SPECIFIC INHIBITOR
OF P-AMYLASE
393
from the reaction mixture occurred rapidly upon seeding with crystals. Enzyme assays. Both (Y- and /3-amylase activities were assayed by the method of Bernfield (8) except that the reactions were carried out in 50 mM phosphate buffer, pH 7.0. The final concentration of soluble starch was 0.5%. Glucoamylase activity was determined by monitoring the release of glucose from starch by the method of Bergmeyer and Bernt (9). Maltase and a-methylglucosidase were assayed by Halvorson’s method (10) as modified by Lai and Axelrod (1). The inhibitor was dissolved in the assay buffer just before use. RESULTS AND DISCUSSION
The maltosylamine, whose synthesis appears not to have been previously reported, melted at 1’78-180”. Anal. calcd. for C,,H,,O,,N: C, 42.23; H, 6.74; N, 4.11. Found: C, 41.89; H, 6.88; N, 4.15. The [(Y];~ was +116.8”. The molar rotation for @maltosylamine, calculated by the method of Isbell and Frush (7), is 43,400 and compares well with the observed value of 43,900. Thus, maltosylamine appears to have the P-configuration at the anomeric carbon, P-Maltosylamine was found to be a relatively effective inhibitor of sweet potato P-amylase. As is seen in Fig. 1, the concentration of maltosylamine required for 50% inhibition is approximately 3 x 10e4M. When the data in Fig. 1 is replotted according to Johnson et al. (11) in the form, log [v, - Vi/Vi] vs log [I] (where v, and vi represent the velocity in the absence and presence of inhibitor, respec-
L
I
02
I
I
04 06 ~ALTOSYLAMINE]
I
OS
tively) the slope is equal to 1.1 (Fig. 2). The slope of the line represents the number of inhibitor molecules binding at each active site. Lineweaver-Burk treatment of the results obtained with a range of inhibitor and substrate concentrations indicates that the inhibition is uncompetitive (Fig. 3). No 1‘nhi bi t ion was observed with glucosylamine at 8.6 mM (3). Maltose, in concentrations up to 1.0 mM, showed only slight inhibition. Sweet potato P-amylase has previously been shown to have an essential sulfhydryl group at or near the active site (12-14). It has been reported that when p-amylase was treated with NEM more than 99% of the activity was rapidly lost, presumably by the alkylation of the most reactive
I IO
mM
1. Inhibition of sweet potato p-amylase by p-maltosylamine. Incubations and assays were as described under Materials and Methods. FIG.
FIG. 2. The data from Fig. 1 was replotted to determine the number of inhibitor molecules bound per active site.
FIG. 3. Inhibition of sweet potato P-amylase by p-maltosylamine. Incubations and assays were as described under Materials and Methods. (0) [I] = 0; (O)[I] = 0.25m~;(A)[I] = &5tbmM;(O)[I] = l.oInM.
394
WALKERANDAXELROD
4.8 x lop4 M, respectively (15, 16). Methyla-D-glucoside has been reported to have a Ki value of 40 mM (2). We found that maltosylamine, at a concentration of approximately 3 x low4 M, inhibited the enzyme by 50% when a 0.5% starch solution was used as substrate. Unlike the previously described instances of inhibition of glycosidases by glycosylamines, maltosylamine appeared to act as an uncompetitive rather than a competitive inhibitor. The results suggest that the substrate must bind to the enzyme first, perhaps eliciting a conformational change in structure, before the inhibitor can bind. However, interpretation of kinetics obtained with a fixed concentration of a somewhat ill-defined substrate such as “soluble starch,” whose components cover a wide range of molecular weights, may be open to question. That the inhibition was directed toward the active site was indicated by the observation that maltosylamine protected the essential sulfhydryl from alkylation by NEM. This thiol group has been reported to be at the active site (12, 14). Maltosylamine appears to be relatively specific for P-amylase. Pancreatic a-amylase and B. subtilis a-amylase (dextrinizing) were not significantly inhibited by maltosylamine. A. niger glucoamylase, an exoglucosidase, was inhibited, but only to a slight degree compared to /3-amylase. Maltase and a-methylglucosidase from S. oviformis were also inhibited by maltosylamine but only at much higher concentrations of inhibitor. Both of these enzymes, as well as glucoamylase, are significantly inhibited with only micromolar concentrations of glucosylamine (1, 3). Thus, the glucoamylase is 50% inhibited by 33 pM glucosylamine when acting on 0.5% soluble starch while the Ki values for maltase and methylglucosidase were 27 and 31 pM, respectively. It is clear from the progress curves of 20 hydrolysis ofp-nitrophenyl-cu-glucoside that \ 0 maltosylamine is a poor inhibitor for t maltase and a-methylglucosidase, and that I I 1 1 I I I 5 IO 15 20 25 it is not appreciably hydrolyzed to glucose TIME (mm) and glucosylamine. Had any significant amounts of glycosylamine been liberated, FIG. 4. Protection of pamylase from NEM inactivathe rate of hydrolysis would have been tion by maltosylamine. The NEM concentration was 50 mM. The maltosylamine concentration was (0) 0; progressively diminished, but such a change (0) 1.0 mM; and (A) 5.0 mix was not seen.
thiol group (14). Similar studies with iodoacetamide (12) showed that cyclohexaamylose, a competitive inhibitor (15, IS), prevented the inactivation suggesting that the essential sulfhydryl is at or near the active site. We found that p-amylase incubated with 50 mM NEM lost 85% of its original activity within 25 min (Fig. 4). The inclusion of as little as 1.0 mM maltosylamine in the reaction mixture substantially protected /3-amylase against this inactivation. In contrast to this, maltose at 5.0 mM afforded no protection. When the NEM was omitted, the enzyme activity was unchanged in the same time period. Maltosylamine at concentrations up to 1.0 mM failed to inhibit c-u-amylase.Glucoamylase, tested in the presence of 0.5% starch, was inhibited 46% by 2.0 mM maltosylamine. a-Amylase (dextrinizing) from B. subtilis was inhibited 16% by a 2.0 UIM maltosylamine concentration. Maltase and a-methylglucosidase from yeast were both inhibited competitively by maltosylamine with Ki values of 14 and 31 mrvr, respectively. p-Amylase catalyzes the hydrolytic removal of p-maltose from the nonreducing end of starch (2). Few inhibitors, other than those which modify protein structure, have been described for this enzyme (5, 17). Cyclohexa- and cycloheptaamylose have been shown to competitively inhibit /3-amylase with Ki values of 5.0 x 10m4 and
MALTOSYLAMINE,
A SPECIFIC
Extensive studies on hen egg white lysozyme have led to the suggestion that the catalytic reaction involves an anionic site, specifically Asp 52, which stabilizes a transient carboxonium ion derived from the substrate (18, 20). Investigations of other glycosidases have also pointed to an essential role for carboxyl groups in the catalytic mechanism (21-23). It has been previously suggested (1) that glycosylamines owe their striking specificity and effectiveness to the possibility that they have a double attraction for the enzyme. They have an amino group which presumably interacts electrostatically with the essential anionic site of the enzyme. They also possess a glycosyl moiety which is specifically adapted to the substrate binding region of the enzyme. It may be presumed that the normal fit of the glycosyl residue directs the amino group precisely into position for the electrostatic interaction. The carboxonium ion hypothesis takes its main support from chemical, kinetic, and structural investigations of lysozyme. This enzyme is known to act with retention of configuration of the glycosyl bond which is hydrolyzed. The possibility of single displacement is therefore ruled out. Studies of deuterium isotope effects support the carboxonium ion hypothesis and rule out the possibility of an S,2 bimolecular displacement (20). All previous demonstrations of glycosylamine inhibition have been made with enzymes that show retention. It is noteworthy that /3-amylase which catalyzes hydrolysis with inversion (5) undergoes specific glycosylamine inhibition. The results presented here suggest that there may be no fundamental difference in the primary mechanism of hydrolysis catalyzed by the two types of enzymes. In fact, Shibaoka et al. (24) have reported that a-amylase (saccharifying) from B. subtilis acts with retention or inversion depending on the pH of the reaction and the nature of the substrate. ACKNOWLEDGMENTS We wish to thank Dr. Ching Tang Yeh for the micro-analyses. We are grateful to Michael H. Whittaker for assistance with some of the enzymatic assays.
INHIBITOR
OF p-AMYLASE
395
REFERENCES 1. LAI, H-Y., AND A~ELROD, B. (1973) Biochem. Biophys. Res. Commun. 54, 463-468. 2. THOMA, J. A., AND KOSHLAND, D. E. (1960) J. Biol. Chem. 235, 2511-2517. 3. LAI, H-Y. (1974) Ph.D. Thesis, Purdue University. 4. WALKER, D. E., AND AXELROD, B. (1978) Arch. Biochem. Biophys. 187, 102-107. 5. THOMA, J. A., SPRADLIN, J. E., AND DYGERT, S. (1971) in The Enzymes (Boyer, P. D., ed.), Vol. V, pp. 115-191, Academic Press, New York. 6. FUKUI, T., AND A~ELROD, B. (1965) Fed. Proc. 24, 220. 7. ISBELL, H. S., AND FRUSH, H. J. (1958) J. Org. Chem. 23, 1309-1319. 8. BERNFIELD, P. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 149- 150, Academic Press, New York. 9. BERGMEYER, H. U., AND BERNT, E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), pp. 1206-1212, Academic Press, New York. 10. HALVORSON, H. (1966) in Methods In Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 8, pp. 559-562, Academic Press, New York. 11. JOHNSON, F. H., EYRING, H., AND WILLIAMS, R. W. (1942) J. Cell. Comp. Physiol. 20, 247-268. 12. THOMA, J. A., AND KOSHLAND, D. E. (1960) J. Mol. Biol. 2, 169-170. 13. THOMA, J. A., KOSHLAND, D. E., SHINKE, R., AND RUSCICA, J. (1965) Biochemistry 4, 714-722. 14. SPRADLIN, J., AND THOMA, J. A. (1970) J. Biol. Chem. 245, 117-127. 15. THOMA, J. A., AND KOSHLAND, D. E. (1960) J. Amer. Chem. Sot. 82, 3329-3333. 16. THOMA, J. A., KOSHLAND, D. E., RUSCICA, J., AND BALDWIN, R. (1963) Biochem. Biophys. Res. Commun. 12, 184-188. 17. FRENCH, D. (1960) in The Enzymes (Boyer, P. D., Lardy, H., AND MYRBACK, K., eds.), Vol. 4, pp. 345-368, Academic Press, New York. 18. BLAKE, C. C. F., JOHNSON, L. N., MAIR, G. A., NORTH, A. C. T., PHILLIPS, D. C., AND SHARMA, V. R. (1967) Proc. Roy. Sot. 167, 378-388. 19. VERNON, C. A. (1967) Proc. Roy. Sot. 167, 389-401. 20. PARSONS, S. M., AND RAFTERY, M. A. (1972) Biochemistry 11, 1623-1629. 21. BAUSE, E., AND LEGLER, G. (1974) 2. Phys. Chem. 355, 438-442. 22. LEGLER, G., AND HARDER, A. (1978) Biochim. Biophys. Acta 524, 102-108. 23. QUARONI, A., AND SEMENZA, G. (1976) J. Biol. Chem. 251, 3250-3253. 24. SHIBAOKA, T., ISHIKURA, K., HIROMI, K., AND WATANABE, T. (1975) J. B&hem. 77,1215-1222.
Maltosylamine, DANIEL Department
a Specific
E. WALKER*
of Biochemistry,
Purdue
Inhibitor1 BERNARD
AND
University,
Received January
of /3-Amylase AXELROD3
West Lafayette,
Indiana
47901
22, 1979
P-Maltosylamine has been synthesized for the first time. It is an effective specific inhibitor of sweet potato /3-amylase. This result extends the observation that I-aminoglycosides are specific inhibitors of exoglycosidases which hydrolyze the corresponding glycose and also demonstrates that an enzyme acting with inversion, as well as those acting with retention of anomeric configuration, can be inhibited by glycosylamines. Maltosylamine, which acts as a reversible inhibitor of P-amylase, appears to be directed to the active site since it protects the essential sulfhydryl group of the enzyme from inactivation by N-ethylmaleimide.
P-amylase acts with inversion (5). We report herein the first recorded synthesis of maltosylamine and a study of its effects on P-amylase.
It has been previously demonstrated that 1-aminoglycosides competitively inhibit the corresponding glycosidase (1). Thus, glycosylamine inhibits glucosidase but not galactosidase, while galactosylamine inhibits galactosidase but not glucosidase. Until the present work, the specific inhibition of glycosidases by their corresponding glycosylamines has been observed only with those enzymes which release monosaccharides. Since it has been rigorously confirmed that P-amylase catalyzes the hydrolytic release of a disaccharide, p-maltose (2), from its natural substrate, cu-1,4-glucan, it was of interest to see if the 1-aminoglycoside of p-maltose would specifically inhibit this enzyme. It was already known that glucosylamine does not significantly inhibit this enzyme (3). It was also of special interest to examine the inhibition of P-amylase by maltosylamine because all previous observations of inhibition by glycosylamines have been made with glycosidases which release their glycosyl product with retention of anomeric configuration (1, 3, 4). It is well known that
MATERIALS
Materials. /%Amylase (sweet potato) and amylo-cu1,4-o-1,6-glucosidase (Aspergillus niger) were from Boehringer-Mannheim GmbH. a-Amylase (hog pancreas) was obtained from P. L. Biochemicals. Calbiochem supplied a-amylase (dextrinizing) from Bacillus subtilis. a-Methylglucosidase from Saccharomyces oviformis was purified by the method of Fukui and Axelrod (6). Maltase from the same organism adapted to maltose was used as a crude extract. N-Ethylmaleimide (NEM)” was obtained from Aldrich Chemical Company. Soluble starch (Lintner’s) was purchased from Fisher Scientific Company. Maltosylamine was Synthesis of maltosylamine. synthesized essentially by the general method which Isbell and Frush (7) have described for the preparation of monoglycosylamines. Ammonium chloride (1.0 g) was dissolved in 50 ml of anhydrous methanol. Maltose (80 g) was added and NH3 was bubbled through the mixture at 0°C until the sugar went into solution. The flask was stoppered and kept in the refrigerator at 4°C for 3 months during which time crystals formed. The product was recrystallized by dissolving in 2 parts HZ0 (40”) and adding 4 parts methanol and 2 parts isopropanol. The crystals which formed at 4” were collected by vacuum filtration and washed with ice-cold methanol saturated with NH, and were dried for 24 h irk oacuo over NaOH. In subsequent preparations, crystallization
* Supported by NIH Research Grant GM 22189. This is Journal Paper No. 7421, Purdue Agricultural Experiment Station. 2 Recipient of U. S. Public Health Predoctoral Trainee Fellowship GM 7211. Present address: Diagnostic Quality Control, Dow Chemical Company, P.O. Box 68511, Indianapolis, Indiana 46268. 3 To who requests for reprints may be sent. 0003-9861/79/080392-04$02.00/O
Copyright 0 1979 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
AND METHODS
4 Abbreviation 392
used: NEM, N-Ethyl
maleimide.
MALTOSYLAMINE.
A SPECIFIC INHIBITOR
OF P-AMYLASE
393
from the reaction mixture occurred rapidly upon seeding with crystals. Enzyme assays. Both (Y- and /3-amylase activities were assayed by the method of Bernfield (8) except that the reactions were carried out in 50 mM phosphate buffer, pH 7.0. The final concentration of soluble starch was 0.5%. Glucoamylase activity was determined by monitoring the release of glucose from starch by the method of Bergmeyer and Bernt (9). Maltase and a-methylglucosidase were assayed by Halvorson’s method (10) as modified by Lai and Axelrod (1). The inhibitor was dissolved in the assay buffer just before use. RESULTS AND DISCUSSION
The maltosylamine, whose synthesis appears not to have been previously reported, melted at 1’78-180”. Anal. calcd. for C,,H,,O,,N: C, 42.23; H, 6.74; N, 4.11. Found: C, 41.89; H, 6.88; N, 4.15. The [(Y];~ was +116.8”. The molar rotation for @maltosylamine, calculated by the method of Isbell and Frush (7), is 43,400 and compares well with the observed value of 43,900. Thus, maltosylamine appears to have the P-configuration at the anomeric carbon, P-Maltosylamine was found to be a relatively effective inhibitor of sweet potato P-amylase. As is seen in Fig. 1, the concentration of maltosylamine required for 50% inhibition is approximately 3 x 10e4M. When the data in Fig. 1 is replotted according to Johnson et al. (11) in the form, log [v, - Vi/Vi] vs log [I] (where v, and vi represent the velocity in the absence and presence of inhibitor, respec-
L
I
02
I
I
04 06 ~ALTOSYLAMINE]
I
OS
tively) the slope is equal to 1.1 (Fig. 2). The slope of the line represents the number of inhibitor molecules binding at each active site. Lineweaver-Burk treatment of the results obtained with a range of inhibitor and substrate concentrations indicates that the inhibition is uncompetitive (Fig. 3). No 1‘nhi bi t ion was observed with glucosylamine at 8.6 mM (3). Maltose, in concentrations up to 1.0 mM, showed only slight inhibition. Sweet potato P-amylase has previously been shown to have an essential sulfhydryl group at or near the active site (12-14). It has been reported that when p-amylase was treated with NEM more than 99% of the activity was rapidly lost, presumably by the alkylation of the most reactive
I IO
mM
1. Inhibition of sweet potato p-amylase by p-maltosylamine. Incubations and assays were as described under Materials and Methods. FIG.
FIG. 2. The data from Fig. 1 was replotted to determine the number of inhibitor molecules bound per active site.
FIG. 3. Inhibition of sweet potato P-amylase by p-maltosylamine. Incubations and assays were as described under Materials and Methods. (0) [I] = 0; (O)[I] = 0.25m~;(A)[I] = &5tbmM;(O)[I] = l.oInM.
394
WALKERANDAXELROD
4.8 x lop4 M, respectively (15, 16). Methyla-D-glucoside has been reported to have a Ki value of 40 mM (2). We found that maltosylamine, at a concentration of approximately 3 x low4 M, inhibited the enzyme by 50% when a 0.5% starch solution was used as substrate. Unlike the previously described instances of inhibition of glycosidases by glycosylamines, maltosylamine appeared to act as an uncompetitive rather than a competitive inhibitor. The results suggest that the substrate must bind to the enzyme first, perhaps eliciting a conformational change in structure, before the inhibitor can bind. However, interpretation of kinetics obtained with a fixed concentration of a somewhat ill-defined substrate such as “soluble starch,” whose components cover a wide range of molecular weights, may be open to question. That the inhibition was directed toward the active site was indicated by the observation that maltosylamine protected the essential sulfhydryl from alkylation by NEM. This thiol group has been reported to be at the active site (12, 14). Maltosylamine appears to be relatively specific for P-amylase. Pancreatic a-amylase and B. subtilis a-amylase (dextrinizing) were not significantly inhibited by maltosylamine. A. niger glucoamylase, an exoglucosidase, was inhibited, but only to a slight degree compared to /3-amylase. Maltase and a-methylglucosidase from S. oviformis were also inhibited by maltosylamine but only at much higher concentrations of inhibitor. Both of these enzymes, as well as glucoamylase, are significantly inhibited with only micromolar concentrations of glucosylamine (1, 3). Thus, the glucoamylase is 50% inhibited by 33 pM glucosylamine when acting on 0.5% soluble starch while the Ki values for maltase and methylglucosidase were 27 and 31 pM, respectively. It is clear from the progress curves of 20 hydrolysis ofp-nitrophenyl-cu-glucoside that \ 0 maltosylamine is a poor inhibitor for t maltase and a-methylglucosidase, and that I I 1 1 I I I 5 IO 15 20 25 it is not appreciably hydrolyzed to glucose TIME (mm) and glucosylamine. Had any significant amounts of glycosylamine been liberated, FIG. 4. Protection of pamylase from NEM inactivathe rate of hydrolysis would have been tion by maltosylamine. The NEM concentration was 50 mM. The maltosylamine concentration was (0) 0; progressively diminished, but such a change (0) 1.0 mM; and (A) 5.0 mix was not seen.
thiol group (14). Similar studies with iodoacetamide (12) showed that cyclohexaamylose, a competitive inhibitor (15, IS), prevented the inactivation suggesting that the essential sulfhydryl is at or near the active site. We found that p-amylase incubated with 50 mM NEM lost 85% of its original activity within 25 min (Fig. 4). The inclusion of as little as 1.0 mM maltosylamine in the reaction mixture substantially protected /3-amylase against this inactivation. In contrast to this, maltose at 5.0 mM afforded no protection. When the NEM was omitted, the enzyme activity was unchanged in the same time period. Maltosylamine at concentrations up to 1.0 mM failed to inhibit c-u-amylase.Glucoamylase, tested in the presence of 0.5% starch, was inhibited 46% by 2.0 mM maltosylamine. a-Amylase (dextrinizing) from B. subtilis was inhibited 16% by a 2.0 UIM maltosylamine concentration. Maltase and a-methylglucosidase from yeast were both inhibited competitively by maltosylamine with Ki values of 14 and 31 mrvr, respectively. p-Amylase catalyzes the hydrolytic removal of p-maltose from the nonreducing end of starch (2). Few inhibitors, other than those which modify protein structure, have been described for this enzyme (5, 17). Cyclohexa- and cycloheptaamylose have been shown to competitively inhibit /3-amylase with Ki values of 5.0 x 10m4 and
MALTOSYLAMINE,
A SPECIFIC
Extensive studies on hen egg white lysozyme have led to the suggestion that the catalytic reaction involves an anionic site, specifically Asp 52, which stabilizes a transient carboxonium ion derived from the substrate (18, 20). Investigations of other glycosidases have also pointed to an essential role for carboxyl groups in the catalytic mechanism (21-23). It has been previously suggested (1) that glycosylamines owe their striking specificity and effectiveness to the possibility that they have a double attraction for the enzyme. They have an amino group which presumably interacts electrostatically with the essential anionic site of the enzyme. They also possess a glycosyl moiety which is specifically adapted to the substrate binding region of the enzyme. It may be presumed that the normal fit of the glycosyl residue directs the amino group precisely into position for the electrostatic interaction. The carboxonium ion hypothesis takes its main support from chemical, kinetic, and structural investigations of lysozyme. This enzyme is known to act with retention of configuration of the glycosyl bond which is hydrolyzed. The possibility of single displacement is therefore ruled out. Studies of deuterium isotope effects support the carboxonium ion hypothesis and rule out the possibility of an S,2 bimolecular displacement (20). All previous demonstrations of glycosylamine inhibition have been made with enzymes that show retention. It is noteworthy that /3-amylase which catalyzes hydrolysis with inversion (5) undergoes specific glycosylamine inhibition. The results presented here suggest that there may be no fundamental difference in the primary mechanism of hydrolysis catalyzed by the two types of enzymes. In fact, Shibaoka et al. (24) have reported that a-amylase (saccharifying) from B. subtilis acts with retention or inversion depending on the pH of the reaction and the nature of the substrate. ACKNOWLEDGMENTS We wish to thank Dr. Ching Tang Yeh for the micro-analyses. We are grateful to Michael H. Whittaker for assistance with some of the enzymatic assays.
INHIBITOR
OF p-AMYLASE
395
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