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[96] α-Glucosidase from yeast
[96]
a-GLUCOSIDASE FROM YEAST
559
triose (I) is almost hydrolyzed as well as pullulan, whereas the tetrasaccharide 62-maltosylmaltose (II) is degraded at a slower rate. o
I
0
0--0
O---O--~ (I)
[96]
O--~ (II)
a-Glucosidase f r o m Yeast 1
By
HARLYN HALVORSON
Maltose 4- H20 ~ 2 glucose
Assay Method
Priraciple. The method is based on the measuring of the continuous release of (PNPG).
p-nitrophenol
from
p-nitrophenol-a-D-glucopyranoside
Reagents Stock solution of PNPG, 0.01 M K Phosphate buffer (M/15) pH 6.8 Enzyme preparation with 100-1000 units/ml
Procedure. A portion (0.1 ml) of enzyme extract is added to a euvette with a 1-cm path length with 2.8 ml of phosphate buffer. After preincubation at 30 °, 0.1 ml of P N P G is added, the solution mixed and incubated at 30 °. Total volume is 3.0 ml. At intervals the optical density of the solution is determined at 400 m~ (EMm 9.6 )K 108). A unit of enzyme is defined as a change in optical density of 0.001/100 seconds with the 3.0 ml reaction mixture. The rates of hydrolysis of other glucosides can be determined by estimating the release of glucose either by the appearance of reducing sugars, ~,8 with a coupled hexokinase-glucose 6-phosphate dehydrogenase system, * or manometrically with a yeast (Torula monosa) incapable of fermenting disaccharides. 5 1The method reported here has been described in Biochim. Biophys. Acta 30, 28 (1958). 2 S. ttestrin and C. C. Lindegren, Arch. Biochem. Biophys. 38, 317 (1952). G. Terui, It. Okada, and Y. Oshima, Technol. Rept. Osaka Univ. 9, 237 (1959). • J. Lamer and C. M. McNickle, J. Biol. Chem. 215, 723 (1955). 5I-I. O. ]~talvorson and S. Spiegelman, J. Bacteriol. 64, 207 (1952).
560
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[96]
Purification Procedure A variety of yeast strains, carrying one or more M genes for ~glucosidase, can be used as a source of enzyme2 The enzymes produced in response to five nonallelic maltose genes are indistinguishable.7 In culturing these yeasts, e.g., Saceharomvces italic~ Y 1125, a nutrient solution of the following composition is employed at pH 5 . 0 : 4 0 1 of water, 200 g peptone (Difeo), 100 g yeast extract (Difco), 80 g (NH4)_~ S04, 100 g KH2P04, 1600 g maltose, 10 g CaCI~, and 10 g MgS04. The cultures of the yeast mass is performed in 100-gallon steel fermenters with strong aeration and stirring. Twelve hours after the addition of a 5% innoculum, maltose is again added to a final concentration of 3%. After 4 hours, the cells are harvested by centrifugation, the fresh cell paste is washed with water and frozen and stored at --20 ° until used. Preparation of Crude Extract. To 200 g cell paste, 20 ml of ethyl acetate was slowly added with stirring. After 30 minutes at room temperature, 300 ml of water was added and the pH was adjusted to 7.0 with NH4OH. The suspension was incubated for 15 hours at room temperature, and the yeast was centrifuged off. The ~-glucosidase is obtained in a cell-free solution. Frgctionation of Enzyme. The extract was frozen at --20 °, stored overnight, and thawed slowly at 3°; the precipitate was removed by centrifugation. The supernatant was dialyzed at 2 ° for 24 hours against M/90 phosphate buffer containing 10-4 M mercaptoethanol. After centrifugation the supernatant (380 ml) was adsorbed with 53 ml of Cad (P04)2 gel (54 mg/ml), the suspension was stirred for 15 minutes, and the gel was removed by centrifugation and washed with cold water. The enzyme was eluted from the gel first with 53 ml of M potassium phosphate, pH 6.8, followed by 24 ml of buffer. The eluates were combined, dialyzed 20 hours against mereaptoethanol phosphate buffer, and readsorbed with Caa(P04)2 gel as above. The enzyme was reeluted with M phosphate buffer, and the eluate was dialyzed as above and concentrated to a small volume by dialysis against M / 5 0 potassium pyrophosphate10-4 mereaptoethanol pH 8.4. The dialyzed enzymes was fractionated by zone eleetrophoresis through insoluble potato starch (see Kunkel and Slater s for details). The enzyme was mixed with dry starch and inserted in a trench cut at 9-10 cm in a 40-cm hemicircular glass trough (5.5 cm I.D.). A potential 6The literature has been reviewed by A. Gottschalk in "The Enzymes" (J. 13. Sumner and K. Myrbiiek, eds.), Vol. I. p. 551. Academic Press, New York, 1950. ' l~I. O. Halvorson, S. Winderman, and J. Gorman, Biochim. Biophys. Acta 67, 42 (1963). 8H. G. Kunkel and R. J. Slater, Proc. Soc. Exptl. Biol. Med. 80, 42 (1952).
[95]
Cz-OLUCOSIDASE FROM YEAST
561
of 140 volts was applied to the electrodes (25 ma) for 18 hours with continuous change of buffer in the electrode chambers. The enzyme band (18-20 cm) was cut and eluted with 20 ml M/15 phosphate buffer, pH 6.8. The overall purification of enzyme is about 100-fold with a 6.1% recovery of enzyme. The fractionation procedure is summarized in the table. FRACTIONATION OF ~-GLucOSIDASE
Step 1. 2. 3. 4. 5.
Autolyzate Frozen-thawed extract First Ca3(PO02 eluate Second Ca3(PO4)2 eluate Zone electrophoresis
Total volume Total units (ml) X 10~ 320 370 77 80 20
91.7 87 52.9 35.3 5.6
Specificactivity (units/ragprotein) 4,100 29,700 44,400 98,000 444,000
Properties
Specificity. The enzyme is specific for a-glucosides2 The enzyme has neither affinity for, nor activity on analogs of a-glucosides which are modified by inversion of hydroxyls on C-2 (mannose) or C-4 (galactose and derivatives), substitution of the hydroxyl on C-6 (raffinose), oxidation of C-6 to C 0 0 H (gluconate) or by substitution on the carboxyls on C-2 to C-6. Elimination of C-6 (methyl-a-D-xylopyranoside) leads to loss of activity, but only reduces affinity for the enzyme (xylose). 1 The loss of affinity with ribose suggests that the hydroxyl on C-3 is required for enzyme activity. Increasing the size of the alkyl group or insertion of an aromatic aglycon progressively increases the velocity constant and decreases the dissociation constant. The enzyme is active on sucrose, turanose and maltose whereas trehalose and isomaltose are complexants but not substrates of the enzyme. Substitution of the 0 of the a-glucoside link by S leads to a loss of substrate activity. Inhibitors. The a-glucosidase of yeast is inhibited by histidine and a number of amines. The inhibition is pH dependent: Tris inhibits more strongly at alkaline than at acid pH. The inhibition at alkaline pH is reversed by excess substrate. As for thiol reagents, a-glucosidase is strongly inhibited by p-mercuribenzoate, iodoacetate, and heavy metal ions. Cysteine reverses the inhibition by PCMB and iodoacetate. Strong inhibition is observed with Cu, Hg, Ag, Pb, and Zn, and moderate inhibition by Fe. These observations point to the existence of an essential thiol group in the enzyme,
562
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[95a]
E]]ect a] pH. The enzyme was fairly stable between pH 6 and 7.8 and exhibits a sharp optimum for activity at pH 6.8 in phosphate buffer. Molecular Weight. The purified enzyme has a sedimentation constant ($2o) of 5.48, leading to a calculated molecular weight of approximately 85,000. Kinetic Properties. Enzymatic hydrolysis of PNPG follows zeroorder kinetics up to 70%, proceeding essentially to quantitative hydrolysis. The reaction is hydrolytic and reversible, although its equilibrium favors hydrolysis.6 The Km for PNPG is 2.0 X 10-4 M. The K~ values of substrates were maltose 3.5 X 10-2 M, sucrose 3.7 X 10-2 M, turanose 1.1 X 10-2M, and a-phenyl glucoside 4 X 10-4M. The following substrates were hydrolyzed in decreasing rates: phenyl-a-D-glucoside, turanose, sucrose, fl-methyl maltoside, PNPG, maltose, butyl-a-D-glueoside, methyl-a-D-glucoside, and ethyl-~-D-glucoside.1 Fructose competes with water for the a-glucosyl units2 The primary product of transglucoylation is isomaltulose. Application to Crude Extracts. Yeasts contain a second enzyme, isomaltase, which shares common substrates, including PNPG, with aglucosidase. The two enzymes can be induced independently. If antisera specific for ~-glucosidase or isomaltase are used, both enzymes can be assayed in mixture. 1° Several other a-glucoside hydrolyzing enzymes have been reported in yeast; however, the relevant enzymes have not been purified and eharacterized2,8,1~ 9G. Avigad, Biochem. J. l, 587 (1959). I°HI. O. Halvorson, H. Okada, and J. Gorman, in "The Cellular Functions of Membrane Transport" (J. F. Hoffman, ed.), p. 171. Prentice-Hall, Englewood Cliffs, New Jersey, 1964. ~1y. Leibowitz, Bull. Res. Council Israel Sect. A, S, 268 (1956).
[ 95a] I s o m a l t a s e f r o m Y e a s t 1 By JOHN GORMAN and HARLYN HALVOtCSON Isomaltose W H20 ~---2 glucose Assay Method Principle. The method is based on the determination of the rate of hydrolysis of p-nitrophenyl-a-D-glucopyranoside (a-PNPG) by isomaltase, employing a spectrophotometric assay of released p-nitrophenol. 1G. Terui, H. Okada, and Y. Oshima, Technol. Rept. Osaka Univ. 9, 237 (1959).
a-GLUCOSIDASE FROM YEAST
559
triose (I) is almost hydrolyzed as well as pullulan, whereas the tetrasaccharide 62-maltosylmaltose (II) is degraded at a slower rate. o
I
0
0--0
O---O--~ (I)
[96]
O--~ (II)
a-Glucosidase f r o m Yeast 1
By
HARLYN HALVORSON
Maltose 4- H20 ~ 2 glucose
Assay Method
Priraciple. The method is based on the measuring of the continuous release of (PNPG).
p-nitrophenol
from
p-nitrophenol-a-D-glucopyranoside
Reagents Stock solution of PNPG, 0.01 M K Phosphate buffer (M/15) pH 6.8 Enzyme preparation with 100-1000 units/ml
Procedure. A portion (0.1 ml) of enzyme extract is added to a euvette with a 1-cm path length with 2.8 ml of phosphate buffer. After preincubation at 30 °, 0.1 ml of P N P G is added, the solution mixed and incubated at 30 °. Total volume is 3.0 ml. At intervals the optical density of the solution is determined at 400 m~ (EMm 9.6 )K 108). A unit of enzyme is defined as a change in optical density of 0.001/100 seconds with the 3.0 ml reaction mixture. The rates of hydrolysis of other glucosides can be determined by estimating the release of glucose either by the appearance of reducing sugars, ~,8 with a coupled hexokinase-glucose 6-phosphate dehydrogenase system, * or manometrically with a yeast (Torula monosa) incapable of fermenting disaccharides. 5 1The method reported here has been described in Biochim. Biophys. Acta 30, 28 (1958). 2 S. ttestrin and C. C. Lindegren, Arch. Biochem. Biophys. 38, 317 (1952). G. Terui, It. Okada, and Y. Oshima, Technol. Rept. Osaka Univ. 9, 237 (1959). • J. Lamer and C. M. McNickle, J. Biol. Chem. 215, 723 (1955). 5I-I. O. ]~talvorson and S. Spiegelman, J. Bacteriol. 64, 207 (1952).
560
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[96]
Purification Procedure A variety of yeast strains, carrying one or more M genes for ~glucosidase, can be used as a source of enzyme2 The enzymes produced in response to five nonallelic maltose genes are indistinguishable.7 In culturing these yeasts, e.g., Saceharomvces italic~ Y 1125, a nutrient solution of the following composition is employed at pH 5 . 0 : 4 0 1 of water, 200 g peptone (Difeo), 100 g yeast extract (Difco), 80 g (NH4)_~ S04, 100 g KH2P04, 1600 g maltose, 10 g CaCI~, and 10 g MgS04. The cultures of the yeast mass is performed in 100-gallon steel fermenters with strong aeration and stirring. Twelve hours after the addition of a 5% innoculum, maltose is again added to a final concentration of 3%. After 4 hours, the cells are harvested by centrifugation, the fresh cell paste is washed with water and frozen and stored at --20 ° until used. Preparation of Crude Extract. To 200 g cell paste, 20 ml of ethyl acetate was slowly added with stirring. After 30 minutes at room temperature, 300 ml of water was added and the pH was adjusted to 7.0 with NH4OH. The suspension was incubated for 15 hours at room temperature, and the yeast was centrifuged off. The ~-glucosidase is obtained in a cell-free solution. Frgctionation of Enzyme. The extract was frozen at --20 °, stored overnight, and thawed slowly at 3°; the precipitate was removed by centrifugation. The supernatant was dialyzed at 2 ° for 24 hours against M/90 phosphate buffer containing 10-4 M mercaptoethanol. After centrifugation the supernatant (380 ml) was adsorbed with 53 ml of Cad (P04)2 gel (54 mg/ml), the suspension was stirred for 15 minutes, and the gel was removed by centrifugation and washed with cold water. The enzyme was eluted from the gel first with 53 ml of M potassium phosphate, pH 6.8, followed by 24 ml of buffer. The eluates were combined, dialyzed 20 hours against mereaptoethanol phosphate buffer, and readsorbed with Caa(P04)2 gel as above. The enzyme was reeluted with M phosphate buffer, and the eluate was dialyzed as above and concentrated to a small volume by dialysis against M / 5 0 potassium pyrophosphate10-4 mereaptoethanol pH 8.4. The dialyzed enzymes was fractionated by zone eleetrophoresis through insoluble potato starch (see Kunkel and Slater s for details). The enzyme was mixed with dry starch and inserted in a trench cut at 9-10 cm in a 40-cm hemicircular glass trough (5.5 cm I.D.). A potential 6The literature has been reviewed by A. Gottschalk in "The Enzymes" (J. 13. Sumner and K. Myrbiiek, eds.), Vol. I. p. 551. Academic Press, New York, 1950. ' l~I. O. Halvorson, S. Winderman, and J. Gorman, Biochim. Biophys. Acta 67, 42 (1963). 8H. G. Kunkel and R. J. Slater, Proc. Soc. Exptl. Biol. Med. 80, 42 (1952).
[95]
Cz-OLUCOSIDASE FROM YEAST
561
of 140 volts was applied to the electrodes (25 ma) for 18 hours with continuous change of buffer in the electrode chambers. The enzyme band (18-20 cm) was cut and eluted with 20 ml M/15 phosphate buffer, pH 6.8. The overall purification of enzyme is about 100-fold with a 6.1% recovery of enzyme. The fractionation procedure is summarized in the table. FRACTIONATION OF ~-GLucOSIDASE
Step 1. 2. 3. 4. 5.
Autolyzate Frozen-thawed extract First Ca3(PO02 eluate Second Ca3(PO4)2 eluate Zone electrophoresis
Total volume Total units (ml) X 10~ 320 370 77 80 20
91.7 87 52.9 35.3 5.6
Specificactivity (units/ragprotein) 4,100 29,700 44,400 98,000 444,000
Properties
Specificity. The enzyme is specific for a-glucosides2 The enzyme has neither affinity for, nor activity on analogs of a-glucosides which are modified by inversion of hydroxyls on C-2 (mannose) or C-4 (galactose and derivatives), substitution of the hydroxyl on C-6 (raffinose), oxidation of C-6 to C 0 0 H (gluconate) or by substitution on the carboxyls on C-2 to C-6. Elimination of C-6 (methyl-a-D-xylopyranoside) leads to loss of activity, but only reduces affinity for the enzyme (xylose). 1 The loss of affinity with ribose suggests that the hydroxyl on C-3 is required for enzyme activity. Increasing the size of the alkyl group or insertion of an aromatic aglycon progressively increases the velocity constant and decreases the dissociation constant. The enzyme is active on sucrose, turanose and maltose whereas trehalose and isomaltose are complexants but not substrates of the enzyme. Substitution of the 0 of the a-glucoside link by S leads to a loss of substrate activity. Inhibitors. The a-glucosidase of yeast is inhibited by histidine and a number of amines. The inhibition is pH dependent: Tris inhibits more strongly at alkaline than at acid pH. The inhibition at alkaline pH is reversed by excess substrate. As for thiol reagents, a-glucosidase is strongly inhibited by p-mercuribenzoate, iodoacetate, and heavy metal ions. Cysteine reverses the inhibition by PCMB and iodoacetate. Strong inhibition is observed with Cu, Hg, Ag, Pb, and Zn, and moderate inhibition by Fe. These observations point to the existence of an essential thiol group in the enzyme,
562
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[95a]
E]]ect a] pH. The enzyme was fairly stable between pH 6 and 7.8 and exhibits a sharp optimum for activity at pH 6.8 in phosphate buffer. Molecular Weight. The purified enzyme has a sedimentation constant ($2o) of 5.48, leading to a calculated molecular weight of approximately 85,000. Kinetic Properties. Enzymatic hydrolysis of PNPG follows zeroorder kinetics up to 70%, proceeding essentially to quantitative hydrolysis. The reaction is hydrolytic and reversible, although its equilibrium favors hydrolysis.6 The Km for PNPG is 2.0 X 10-4 M. The K~ values of substrates were maltose 3.5 X 10-2 M, sucrose 3.7 X 10-2 M, turanose 1.1 X 10-2M, and a-phenyl glucoside 4 X 10-4M. The following substrates were hydrolyzed in decreasing rates: phenyl-a-D-glucoside, turanose, sucrose, fl-methyl maltoside, PNPG, maltose, butyl-a-D-glueoside, methyl-a-D-glucoside, and ethyl-~-D-glucoside.1 Fructose competes with water for the a-glucosyl units2 The primary product of transglucoylation is isomaltulose. Application to Crude Extracts. Yeasts contain a second enzyme, isomaltase, which shares common substrates, including PNPG, with aglucosidase. The two enzymes can be induced independently. If antisera specific for ~-glucosidase or isomaltase are used, both enzymes can be assayed in mixture. 1° Several other a-glucoside hydrolyzing enzymes have been reported in yeast; however, the relevant enzymes have not been purified and eharacterized2,8,1~ 9G. Avigad, Biochem. J. l, 587 (1959). I°HI. O. Halvorson, H. Okada, and J. Gorman, in "The Cellular Functions of Membrane Transport" (J. F. Hoffman, ed.), p. 171. Prentice-Hall, Englewood Cliffs, New Jersey, 1964. ~1y. Leibowitz, Bull. Res. Council Israel Sect. A, S, 268 (1956).
[ 95a] I s o m a l t a s e f r o m Y e a s t 1 By JOHN GORMAN and HARLYN HALVOtCSON Isomaltose W H20 ~---2 glucose Assay Method Principle. The method is based on the determination of the rate of hydrolysis of p-nitrophenyl-a-D-glucopyranoside (a-PNPG) by isomaltase, employing a spectrophotometric assay of released p-nitrophenol. 1G. Terui, H. Okada, and Y. Oshima, Technol. Rept. Osaka Univ. 9, 237 (1959).