/ . Biochem., 77, 695-703 (1975)
Studies on the Subsite Structure of Amylases I. Interaction of Glucoamylase1 with Substrate and Analogues Studied by Difference-spectrophotometry1 Masatake OHNISHI, Hisashi KEGAI, and Keitaro HIROMI Department of Food Science and Technology, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, November 2, 1974
Studies were made on the ultraviolet difference-spectra of glucoamylase from Rhizopus niveus [EC 3.2.1.3] specifically produced by the substrate maltose and the inhibitors, glucose, glucono-1:5-lactone (gluconolactone), methyl /9-D-glucoside, cellobiose, and cyclohexa-, and cyclohepta-amyloses. Of these, maltose and gluconolactone produced characteristic difference spectra with a trough near 300 nm. Based on studies with a model compound for a tryptophan residue, Ac-Trp, this trough was attributed to the effect of a negative charge upon the tryptophan residue. From the concentration dependency of the difference spectra, the dissociation constants of the complexes between the enzyme and maltose, glucose, and gluconolactone were evaluated to be 1.2 mM, 51 mM, and 1.5 mM, respectively. These values are in good agreement with the values of Km or Ki obtained from the steady-state kinetics. The difference-spectrophotometric data suggested that referring to the values of subsite affinities of glucoamylase, maltose, and gluconolactone occupy mainly Subsite 1, where the nonreducing-end glucose residue of a substrate is bound in a productive form and that a tryptophan residue which shows a trough near 300 nm in difference spectra is located in this subsite.
Difference-spectrophotometric investigations on the interaction of an enzyme with substrates or substrate analogues provides valuable information on the structure of the active site. For example, from studies on the specific interactions of lysozyme [EC 3. 2.1.17] and amy, ,„_ „ „— , 1 EC 3.2.1.3], o - l , 4 : 1,6-Glucan-4:6-glucohydrolase. ,1 „ , . . , . . _ , t, This study was supported in part by a grant from the Ministry of Education. Abbreviations: Gluconolactone, glucono-1:5-lactone; Ac-Trp-OEt, N-Acetyl-L-tryptophan ethyl ester; AcTyr-OEt, N-Acetyl-L-tyrosine ethyl ester; Ac-Trp, N-Acetyltryptophan. Vol. 77, No. 4, 1975
lases with their substrates or analogues tryptophan and/or tyrosine residues have been located in the certain subsite of these enzymes (1—6). The slowly-reacting substrates or substrate analogues, mono- or oligo-saccharides are best suited for these studies. The subsite structure, i.e., the number of . . , , t i . - i . a i subsites and the arrangement of .subsite affini,. 7 , , ,. , , ties ' h a s ***•* s t u d i e d f o r s e V e r a l k l n d s ° f amylase by the kinetic method ( 7 - / 2 ) and also by product analysis (13). Besides these methods, difference-spectrophotometry of enzyme proteins produced by
695
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M. OHNISHI, H. KEGAI, and K. HIROMI
696
substrates and analogues has proved a useful probe of the subsite structure of amylases. In this way, the location and number of tryptophan and/or tyrosine residues located at the subsite(s) to which the substrate or analogue may bind, have been estimated for various amylases (3—5). In this paper, we studied difference spectra due to the tryptophan and tyrosine residues of glucoamylase from Rhizopus niveus in the presence of a substrate(maltose) and several analogues(glucose, gluconolactone, methyl /9-glucoside, cellobiose, and cyclohexa-, and cyclohepta-amyloses). A characteristic differencespectrum with a trough around 300 nm due to a tryptophan residue was found to be produced only by the substrate maltose and one of the inhibitors, gluconolactone, which may be regarded as a transition state analogue. This tryptophan residue seems to be located in the vicinity of the catalytic site of the enzyme. EXPERIMENTAL Material — Crystalline glucoamylase from Rhizopus niveus (pure grade) was purchased from Seikagaku Kogyo Co., Ltd. and used without further purification. Maltose, and cyclohexa-, and cyclohepta-amyloses were products of Hayashibara Biochemical Laboratories. Glucose, methyl /3-D-glucoside, cellobiose, glucono-1 : 5-lactone (gluconolactone), the ethyl esters of N-acetyl-L-tryptophan and N-acetylL-tyrosine, N-acetyl-L-tryptophan, and all other reagents and solvents of guaranteed grade were purchased from Nakarai Chemicals, Ltd. Hydrolysis of gluconolactone during measurements of difference spectra was less than 15% under the experimental conditions employed. Measurements of Difference Spectra—difference spectra of glucoamylase, the ethyl esters of N-acetyl-L-tryptophan (Ac-Trp-OEt) and N-acetyl-L-tyrosine (Ac-Tyr-OEt), and Nacetyltryptophan (Ac-Trp) were measured with a Shimadzu UV-200 Recording Spectrophotometer (0.1 absorbance full scale) as described elsewhere (3) or a Union Giken High Sensitivity Spectrophotometer SM-401 (0.02 absorbance full scale) at pH 4.5 and 25°. For the substrate maltose, which is hydrolyzed at an
appreciable speed, measurements were completed within 18 sec after mixing the enzyme and substrate solution using the Spectrophotometer SM-401 and a manual mixing apparatus (Union Giken RA-101). Less than 16% of the maltose was hydrolyzed during the measurements. Absorbances at 280 nm of the enzyme, Ac-Trp-OEt, Ac-Tyr-OEt, and Ac-Trp were kept below 1.0 to avoid possible error due to stray light. The concentration of the enzyme was determined spectrophotometrically at 280 nm taking the absorption of 1% solution as 13.6 cm" 1 and the molecular weight as 58,000, as determined by SDS gel electrophoresis (Tsujisaka, Y., personal communication). The molecular absorption coefficients of AcTrp-OEt and Ac-Tyr-OEt at 282 nm were taken as 5,550 and 1,340, respectively (14). Amino Acid Analysis of the Enzyme— Amino acid analysis was performed with an amino acid analyzer (Hitachi model KLA-3) (75, 16). Enzyme samples were hydrolyzed in 6 M HC1 for 22 hr at 115°. Tryptophan residues were determined using />-dimethylaminobenzaldehyde (16). RESULTS Difference Spectra of Glucoamylase Produced by Maltose, Glucose, and Gluconolactone —Figures 1-a and b show typical examples of the difference spectra of the glucoamylase produced by 1.8 mM and 139 mM maltose, respectively. The spectra were recorded within 18 sec after mixing the enzyme and maltose. Measurement of the difference spectrum produced with such a low concentration (1.8 mM) of substrate was achieved with the High Sensitivity Spectrophotometer and the manual mixing apparatus. Less than 6% of the maltose was hydrolyzed by the enzyme during this measurement. The difference spectra have peaks at 285 nm and 292 nm and a characteristic trough at 300—302 nm, which disappears as maltose is hydrolyzed. The binding of gluconolactone was found to produce a difference spectrum with peaks at 285 nm and 292 nm, and a characteristic trough at 301 nm, which is quite similar to that produced by maltose, as shown in Fig. 2. However, the / . Biochem.
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INTERACTION OF GLUCOAMYLASE WITH SUBSTRATE AND ANALOGUES
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280 *»0 300 WAVELENSTH (am)
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280 300 WAVELENGTH (nm)
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Fig. 3. T h e difference spectrum of glucoamylase produced by glucose. Enzyme, 7.7 ftM; Glucose, (a) 0.96 M ; (b) 0 M. T h e difference spectrum was recorded with a Recording Spectrophotometer (Shimadzu UV-200).
2500 -
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260 2*0 300 WAVELEMOTH tea)
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Fig. 1. The difference spectra of glucoamylase produced by maltose. Enzyme, 11.5 (IM ; Maltose, (a) 1.8 mM; (b) 139 DM ; The difference spectra were recorded from 6 to 18 sec (scanning speed 5 nm/sec) after mixing the enzyme and the substrate maltose using a High Sensitivity Spectrophotometer (Union Giken SM-401) and a mixing apparatus (Union Giken RA-101). MALTOSE (%l Fig. 4. Effect of the concentration of maltose upon the absorptivity difference of glucoamylase observed at 285, 292, and 302 n m . • , J £ a p p a t 285 n m ; O , 4£app a t 292 n m ; •, J£app at 302 n m ; The straight lines a and b represent the nonspecific solvent perturbation effects of maltose \ses text) at 292 n m and at 285 nm, respectively.
260
320 340 280 300 WAVELENGTH (run) Fig. 2. T h e difference spectrum of glucoamylase produced by gluconolactone. Enzyme, 14 fiM; Gluconolactone, 5.8 mM. T h e difference spectrum w a s recorded with a Recording Spectrophotometer (Shima
Studies on the Subsite Structure of Amylases I. Interaction of Glucoamylase1 with Substrate and Analogues Studied by Difference-spectrophotometry1 Masatake OHNISHI, Hisashi KEGAI, and Keitaro HIROMI Department of Food Science and Technology, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, November 2, 1974
Studies were made on the ultraviolet difference-spectra of glucoamylase from Rhizopus niveus [EC 3.2.1.3] specifically produced by the substrate maltose and the inhibitors, glucose, glucono-1:5-lactone (gluconolactone), methyl /9-D-glucoside, cellobiose, and cyclohexa-, and cyclohepta-amyloses. Of these, maltose and gluconolactone produced characteristic difference spectra with a trough near 300 nm. Based on studies with a model compound for a tryptophan residue, Ac-Trp, this trough was attributed to the effect of a negative charge upon the tryptophan residue. From the concentration dependency of the difference spectra, the dissociation constants of the complexes between the enzyme and maltose, glucose, and gluconolactone were evaluated to be 1.2 mM, 51 mM, and 1.5 mM, respectively. These values are in good agreement with the values of Km or Ki obtained from the steady-state kinetics. The difference-spectrophotometric data suggested that referring to the values of subsite affinities of glucoamylase, maltose, and gluconolactone occupy mainly Subsite 1, where the nonreducing-end glucose residue of a substrate is bound in a productive form and that a tryptophan residue which shows a trough near 300 nm in difference spectra is located in this subsite.
Difference-spectrophotometric investigations on the interaction of an enzyme with substrates or substrate analogues provides valuable information on the structure of the active site. For example, from studies on the specific interactions of lysozyme [EC 3. 2.1.17] and amy, ,„_ „ „— , 1 EC 3.2.1.3], o - l , 4 : 1,6-Glucan-4:6-glucohydrolase. ,1 „ , . . , . . _ , t, This study was supported in part by a grant from the Ministry of Education. Abbreviations: Gluconolactone, glucono-1:5-lactone; Ac-Trp-OEt, N-Acetyl-L-tryptophan ethyl ester; AcTyr-OEt, N-Acetyl-L-tyrosine ethyl ester; Ac-Trp, N-Acetyltryptophan. Vol. 77, No. 4, 1975
lases with their substrates or analogues tryptophan and/or tyrosine residues have been located in the certain subsite of these enzymes (1—6). The slowly-reacting substrates or substrate analogues, mono- or oligo-saccharides are best suited for these studies. The subsite structure, i.e., the number of . . , , t i . - i . a i subsites and the arrangement of .subsite affini,. 7 , , ,. , , ties ' h a s ***•* s t u d i e d f o r s e V e r a l k l n d s ° f amylase by the kinetic method ( 7 - / 2 ) and also by product analysis (13). Besides these methods, difference-spectrophotometry of enzyme proteins produced by
695
Downloaded from https://academic.oup.com/jb/article-abstract/77/4/695/2185225 by University of California Santa Barbara/Davidson Library user on 10 March 2018
M. OHNISHI, H. KEGAI, and K. HIROMI
696
substrates and analogues has proved a useful probe of the subsite structure of amylases. In this way, the location and number of tryptophan and/or tyrosine residues located at the subsite(s) to which the substrate or analogue may bind, have been estimated for various amylases (3—5). In this paper, we studied difference spectra due to the tryptophan and tyrosine residues of glucoamylase from Rhizopus niveus in the presence of a substrate(maltose) and several analogues(glucose, gluconolactone, methyl /9-glucoside, cellobiose, and cyclohexa-, and cyclohepta-amyloses). A characteristic differencespectrum with a trough around 300 nm due to a tryptophan residue was found to be produced only by the substrate maltose and one of the inhibitors, gluconolactone, which may be regarded as a transition state analogue. This tryptophan residue seems to be located in the vicinity of the catalytic site of the enzyme. EXPERIMENTAL Material — Crystalline glucoamylase from Rhizopus niveus (pure grade) was purchased from Seikagaku Kogyo Co., Ltd. and used without further purification. Maltose, and cyclohexa-, and cyclohepta-amyloses were products of Hayashibara Biochemical Laboratories. Glucose, methyl /3-D-glucoside, cellobiose, glucono-1 : 5-lactone (gluconolactone), the ethyl esters of N-acetyl-L-tryptophan and N-acetylL-tyrosine, N-acetyl-L-tryptophan, and all other reagents and solvents of guaranteed grade were purchased from Nakarai Chemicals, Ltd. Hydrolysis of gluconolactone during measurements of difference spectra was less than 15% under the experimental conditions employed. Measurements of Difference Spectra—difference spectra of glucoamylase, the ethyl esters of N-acetyl-L-tryptophan (Ac-Trp-OEt) and N-acetyl-L-tyrosine (Ac-Tyr-OEt), and Nacetyltryptophan (Ac-Trp) were measured with a Shimadzu UV-200 Recording Spectrophotometer (0.1 absorbance full scale) as described elsewhere (3) or a Union Giken High Sensitivity Spectrophotometer SM-401 (0.02 absorbance full scale) at pH 4.5 and 25°. For the substrate maltose, which is hydrolyzed at an
appreciable speed, measurements were completed within 18 sec after mixing the enzyme and substrate solution using the Spectrophotometer SM-401 and a manual mixing apparatus (Union Giken RA-101). Less than 16% of the maltose was hydrolyzed during the measurements. Absorbances at 280 nm of the enzyme, Ac-Trp-OEt, Ac-Tyr-OEt, and Ac-Trp were kept below 1.0 to avoid possible error due to stray light. The concentration of the enzyme was determined spectrophotometrically at 280 nm taking the absorption of 1% solution as 13.6 cm" 1 and the molecular weight as 58,000, as determined by SDS gel electrophoresis (Tsujisaka, Y., personal communication). The molecular absorption coefficients of AcTrp-OEt and Ac-Tyr-OEt at 282 nm were taken as 5,550 and 1,340, respectively (14). Amino Acid Analysis of the Enzyme— Amino acid analysis was performed with an amino acid analyzer (Hitachi model KLA-3) (75, 16). Enzyme samples were hydrolyzed in 6 M HC1 for 22 hr at 115°. Tryptophan residues were determined using />-dimethylaminobenzaldehyde (16). RESULTS Difference Spectra of Glucoamylase Produced by Maltose, Glucose, and Gluconolactone —Figures 1-a and b show typical examples of the difference spectra of the glucoamylase produced by 1.8 mM and 139 mM maltose, respectively. The spectra were recorded within 18 sec after mixing the enzyme and maltose. Measurement of the difference spectrum produced with such a low concentration (1.8 mM) of substrate was achieved with the High Sensitivity Spectrophotometer and the manual mixing apparatus. Less than 6% of the maltose was hydrolyzed by the enzyme during this measurement. The difference spectra have peaks at 285 nm and 292 nm and a characteristic trough at 300—302 nm, which disappears as maltose is hydrolyzed. The binding of gluconolactone was found to produce a difference spectrum with peaks at 285 nm and 292 nm, and a characteristic trough at 301 nm, which is quite similar to that produced by maltose, as shown in Fig. 2. However, the / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/77/4/695/2185225 by University of California Santa Barbara/Davidson Library user on 10 March 2018
697
INTERACTION OF GLUCOAMYLASE WITH SUBSTRATE AND ANALOGUES
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280 *»0 300 WAVELENSTH (am)
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280 300 WAVELENGTH (nm)
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Fig. 3. T h e difference spectrum of glucoamylase produced by glucose. Enzyme, 7.7 ftM; Glucose, (a) 0.96 M ; (b) 0 M. T h e difference spectrum was recorded with a Recording Spectrophotometer (Shimadzu UV-200).
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260 2*0 300 WAVELEMOTH tea)
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Fig. 1. The difference spectra of glucoamylase produced by maltose. Enzyme, 11.5 (IM ; Maltose, (a) 1.8 mM; (b) 139 DM ; The difference spectra were recorded from 6 to 18 sec (scanning speed 5 nm/sec) after mixing the enzyme and the substrate maltose using a High Sensitivity Spectrophotometer (Union Giken SM-401) and a mixing apparatus (Union Giken RA-101). MALTOSE (%l Fig. 4. Effect of the concentration of maltose upon the absorptivity difference of glucoamylase observed at 285, 292, and 302 n m . • , J £ a p p a t 285 n m ; O , 4£app a t 292 n m ; •, J£app at 302 n m ; The straight lines a and b represent the nonspecific solvent perturbation effects of maltose \ses text) at 292 n m and at 285 nm, respectively.
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320 340 280 300 WAVELENGTH (run) Fig. 2. T h e difference spectrum of glucoamylase produced by gluconolactone. Enzyme, 14 fiM; Gluconolactone, 5.8 mM. T h e difference spectrum w a s recorded with a Recording Spectrophotometer (Shima
