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Glucosamine degradation by Escherichia coli. III. Isolation and studies of “phosphoglucosaminisomerase”
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
66, 333-339 (1957)
Glucosamine Degradation by Escherichia Coll. III. Isolation and Studies of "Phosphoglucosaminisomerase"~ Jack B. Wolfe 2, Barbara B. Britton and Henry I. Nakada From the Scripps Clinic and Research Foundation, La Jolla, California Received May 28, 1956 INTRODUCTION
Previous work in this and other laboratories have demonstrated that a n e n z y m e s y s t e m f r o m Escherichia coli is c a p a b l e of d e a m i n a t i n g gluc o s a m i n e - 6 - P Q (1-3). F u r t h e r , evidence i n d i c a t i n g a n isomeric conv e r s i o n of glucosamine-6-PO4 to f r u c t o s e - 6 - P Q a n d a m m o n i a h a s been p r e s e n t e d (2). I n these earlier r e p o r t s , however, t h e n a t u r e of t h e enz y m e s y s t e m concerned w i t h this c o n v e r s i o n was n o t e l u c i d a t e d . T h e p r e s e n t c o m m u n i c a t i o n p r e s e n t s d a t a on t h e i s o l a t i o n a n d f u r t h e r p u r i f i c a t i o n of t h e e n z y m e s y s t e m " p h o s p h o g l u c o s a m i n i s o m e r a s e " a n d s t u d i e s on some of its p r o p e r t i e s . METHODS
Determination of glueose6-PO4 was carried out by the use of Zwischenferment obtained from Armour and Company and further purified by acid precipitation at p g 4.4 as described by Kornberg (4). The final product was free of phosphoglucoisomerase as indicated by its failure to form ketose from glucose-6-PO4 under the experimental conditions employed. Ascending paper chromatographic analysis was carried out using conventional procedures. The synthesis of glucosamine6-PO4 and the method for determination of ammonia and ketose have been described previously (2). The reported values in all cases are corrected for substrate and enzyme controls incubated independently during the course of the reaction. The experimental conditions are reported in the legends of the text. 1 This investigation was supported in part by Research Grant #291 (C2) from the Division of Research Grants and Fellowships of the National Institutes of Health, U. S. Public Health Service. Charles Willard Stimson Postdoctoral Fellow in Biochemistry. Present address: Department of Medical Microbiology, University of Southern California, Los Angeles, California. 333
334
J . B . W O L F E ~ B . B . B R I T T O I ~ A N D H . I . NAKKDA_
TABLE I Phosphoglucoisomerase and " Phosphoglucosaminisomerase" Activities before and after Ca~(P04)2 Gel Treatment The reaction mixture contained: 0.5 ml. enzyme, 4.4 umoles glucosamine-6PO4, and 0.01M phosphate buffer pH 7.5 to a total volume of 1.0 ml. Incubation 24°, air atmosphere. Preparation
First purification b Supernatant after addition ~ ml, Ca3(PO~)2 (14.5 mg. dry wt,) Supernatant after second addition of ~ ml. Ca3(PO4)2 Supernatant from third addition of Ca3(PO~)2 (~ ml.) Elution of third Ca3(P0,)2 adsorption with pit 7.0 P0, buffer (0.01 M)
Specific activitya of Glucosamine Glucose-5-PO~ 5-P04
142 189.5
151 162.5
205 24.1 2390
54.3 0 0
a Micrograms of ketose/min./mg, protein N. b Partially purified preparation [see Ref. (2)]. Purification of "Phosphoglucosaminisomerase" Glucosamine-grown lyophilized cells of Escherichia coli were extracted and partially purified as previously described (2).3 T o a given volume of the partially purified extract (pH 6.0) containing 11.9 rag. protein in 20 ml. was added small increments of calcium phosphate gel (dry weight 29 mg./ml.). The gel was removed b y centrifugation, and the supernatant fluid was tested for its ability to convert glucosamine-6-PQ or glucose-6-PO4 to ketose. Table I presents typical data obtained b y this procedure. I£ can be seen that phosphoglucoisomerase was more readily adsorbed b y the calcium phosphate gel than "phosphoglucosaminisomerase." Elution 'of the third calcium phosphate gel precipitate with 10 ml. of 0.01 M phosphate buffer (pH 7.0) resulted in a highly purified phosphoglucosaminisomerase preparation that was free of phosphoglucoisomerase activity. As shown in Table I, the partially purified preparation, after fractional calcium phosphate gel adsorption and elution, was further purified 17-fold. This preparation does not form ketose from acetylglucosamine-6-PO~. The preparation which represented an approx3 In contrast to glucose-grown cells, the specific activity of the partially purified preparation toward glucosamine 6-phosphate and glucose 6-phosphate were 142 and 151, respectively. These values represent a 17-fold increase in deaminating activity and an 18-fold decrease in phosphoglucoisomerase activity when compared with the partially purified preparation obtained from glucose-grown cells.
GLUCOSAMINE DEGRADATION. III
335
TABLE II Relationship of Fructose Formation to Ammonia Production during Glucosamine-6-P04 Deamination
The reaction mixture contained: enzyme (11.1 ~g. prot. N.), 11.3 ~moles glucosamine-6-PO4,0.01 M phosphate buffer, pit 7.5, to a total volume of 2.7 ml. Incubation 24°, air atmosphere. Time, rain.
Exp~i~ent Productdetermined 1 Ammonia, ~moles Fructose, ~moles 2 Ammonia, ~moles Fructose, t,moles
20 4.3 3.8
40 5.3 5.8
60 7.1 7.0
80 8.2 8.5
3.8
5.4
7.0
8.3
3.5
5.3
7.9
8.7
120 10.1 10.5 9.9 10.3
200 10.9 11.2 10.7 11.0
imately 150-fold purification of the glueosamine-6-PO4 deamination system was the one used for further studies. Table I I gives ketose and ammonia values obtained during the degradation of 11.3 ~moles of glucosamine-6-PO4 b y the highly purified preparation. The results indicate that approximately equivalent amounts of ammonia and fruetose-6-PO~ are produced during the deamination of glueosamine-6-PO4. These results conclusively show that a ketose is the initial product of glucosamine-6-PO4 deamination. Approximately 95-100 % of the added ghicosamine-6-P04 can be accounted for as ketose at the completion of the reaction (Table II). This would indicate that the reaction is far to the right and, in a physiological sense, is probably irreversible. The effect of p H on ketose formation from glucosamine-6-P04 was determined. The results given in Table I I I indicate that the highly purified "phosphoglucosaminisomerase" was active over a broad p H range with a maximum of ketose formation at approximately 7.8. These results a r e quite different from that reported with the partially purified preparation obtained from glucose-grown cells (2). One possible explanation for the variance in p H optima for ketose formation between preparations could be due to a greater acid sensitivity of the highly active phosphoghicoisomerase present in the partially purified preparation. Thus, in the presence of glucosamine-6-PO4, which inhibits phosphoglucoisomerase, an enhanced inhibition of fruetose-6-PO4 removal to glucose-6-P04 might occur under acid conditions. Product Analysis
From our present knowledge of the enzyme system responsible for the deamiaation of glucosamine-6-PO4, the products appear to be ammonia and a ketose
336
J. B. WOLFE~ B. B. B R I T T O N AND H. I. N A I ~ D A
TABLE I I I
Effect of pH on Rate of Deamination of Glucosamine-6-P04 by Purified "Phosphoglucosaminisomerase" Reaction mixture contained: 0.25 ml. of enzyme (2.4 t~g. prof. N.), 4.4 #moles glucosamine-6-PO4,0.01 M phosphate buffer at the indicated ptt in a total volume of 1.0 ml. Incubation 24 °, air atmosphere. The reported pH values were determined at the end of the experimental period. pH
Fruct0se-6-P04 lS rain. 30 rain. ~mole ttmole
5.3 6.1 7.1 7.5 7.8 8.3 8.8
0.164 0.233 0.278 0.298 0.321 0.300 0.190
0.239 0.389 0.57 0.63 0.68 0.60 0.385
which is probably fructose-6-PO4 (2, 3). Evidence that the ketose formed is fructose-6-PO~ is summarized below. The reaction products obtained at the completion of the experiment shown in Table II were used for analysis. Employing the Elson and Morgan colerimetric method, only trace amounts of glueosamine could be detected. An aliquot of this reaction product containing 1.5 t~meles of ketose was tested for the presence of glucose-6-PO4 by the addition of triphosphopyridine nucleotide (TPN) and Zwisehenferment (which catalyzed the oxidation of glucose-6-PO4 to 6-phosphoglueonate with T P N as the cofactor). As shown in Table IV, only a negligible amount of glucose-6-PO4 was detectable. However, the addition of purified phosphoglueoisomerase (which catalyzes the interconversion of glucose6-PO4 and fructose-6-PO4) to this system converted the ketose to glucose-6-PO4 , which was then oxidized by the TPN-Zwischenferment system. Thus, the product appears to be fructose-6-PO4. TABLE IV
Product Analysis: Enzymatic Evidence for Fructose-6-P04 Reaction mixture contained: 0.5 ml. of 0.04 M glycylglycine buffer pI-I 7.5, 20 amoles MgC12,1.2 ~moles triphosphopyridine nucleotide, 0.1 rag. purified Zwischenferment, and an aliquot of the reaction mixture containing 1.5 ~moles ketose in a total volume of 2.7 ml. After the initial glucose-6-PO~ content was determined, 1 rag. of phosphoglucoisomerase was added for fructose-6-PO4 determinations. Treatment
Ketose ~moles
Glucose-6-PO4 ,u~oles
Untreated Zwischenferment Phosphoglucoisomerase -~ Zwischenferment
1.5 1.5 0.14
0.05 1.16
337
GLUCOSAiVIINE DEGR&DATIONo III
TABLE V
Product Analysis: Paper Chromatographic Evidence for Fructose-6-P04 RI
Substrate
Rf ButPhenol HAcH20
Color obtained with various reagents =~-Anisidlne
Ninhydrin
Molybdate
Reaction mixture Fructose-6-PO4 Glucose-6-PO~ Glucosamine-6-P04
0.13 0.13 0.17 0.14
0.08 0.08 0.11 0.12
Lemon-yellow Lemon-yellow Brown Brown
No color No color No color Blue
Blue
Reaction mixture after treatment with phosphatase Fructose Glucose Glucosamine. HC1
O.55 0.25
Lemon-yellow
No color
No color
0.55 0.25 0.41 0.20 0.2310.16
Lemon-yellow Brown Light brown
No color No color Blue
No color No color No color
i
Blue Blue Blue
Identification of the product as fructose-6-PO4 was obtained by paper chromatographic studies (Table V). Using the unidimensional ascending method, the original reaction mixture was chromatographed, using phenol (5) or butanolacetic acid-water (6). In every instance, only a single spot was detectable. Since the Ri values of glucosc-6-PO4, fructose-6-P04, and glucosamine-6-PO4 are relatively close together, further identification was accomplished by different color tests. The unknown spot was lemon-yellow by p-anisidine reagent (7), as was fructose-6-PQ, whereas glucose-6-PO4 gave a brown color. Further, treatment with Hanes-Isherwood phosphate reagent (8) gave a single blue spot that corresponded to the yellow fructose-6-PO4 spot given by the p-anisidine reagent. A separate ehromatogram was also sprayed with ninhydrin reagent. Known glucosaminc-6-PO4 in amounts of less than 1 ~g. was detectable by ninhydrin, but no ninhydrin-reactive spots could be shown on a chromatogram of the reaction mixture. Further identification of the ffuctose-6-P04 was accomplished by treating an aliquot of the reaction mixture with intestinal phosphatase (which was tested and found to be free of phosphoglucoisomerase activity). Chromatography of the phosphatase-treated material gave a single spot whose RI value was identical with fructose and whose color development with p-anisidine gave a yellow color identical with that of known fructose. These data indicate that fructose-6-PO4 is a primary reaction product of phosphoglucosaminisomerase.
Evidence for the Participation of a Sulfhydryl Group Initial experiments with p-ehloromercuribenzoate indicated that a concentration of 10-3 M completely inhibited deamination of glucosamine-6-PO4 by "phosphoglucosaminisomerase." This would indicate
338
ft. B. WOLFE~ B. B. BRITTON AND H. I. NAKADA
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60 90 MINUTES F~G. 1. Inhibition of glucosamine-6-PO4 deamination by p-chloromercuribenzoate. Reaction mixture contained: Enzyme (1.4 ~g. prof. N.), 4.4 ~moles glucosamine6-PO4,0.01 M phosphate buffer pH 7.5, and, where indicated, 10-4 M p-chloromercuribenzoate in a total volume of 0.75 ml. At the time indicated, 0.1 ml. of 0.1 M reduced glutathione was added. Incubation 24°, air atmosphere. )<--no inhibitor. O--p-chloromercuribenzoate added.
that a sulfhydryl group was involved in the reaction (9). To substantiate this finding further, glueosamine-6-PO4 was incubated with "phosphoglucosaminisomerase" in the presence and absence of 10-~ M pehloromercuribenzoate. After 45 rain. incubation, 10-2 M glutathione was added to both reaction vessels. The results given in Fig. 1 indicate that prior to the addition of 10-3 M glutathione, the rate of glucosamine6-PO~ deamination was drastically inhibited by the presence of pchloromercuribenzoate. The addition of 10 ~moles glutathione, however, reversed the inhibition, for an increase in rate of deamination was then observed. These results indicate that sulfhydryl groups are involved in the deamination of glucosamine-6-PO4 by these preparations. The data presented clearly demonstrate that fructose-6-PO4 and ammonia are the products of an enzymatic isomerization of glucosalnine6-PO~ by an enzyme system provisionally called "phosphoglucosaminisomerase." From chemical considerations, there appear to be two steps involved in the reaction: (a) isomerization to a compound resembling 2-iminofructose-6-PO4 i and (b) a hydrolytic deamination of this postulated intermediate to form fructose-6-PO4 and ammonia. In this respect the mechanism of deamination would appear to be dependent upon an
GLUCOSAMIRTE DEGRADATION'. III
339
intramolecular oxidation and reduction taking place between carbon atoms 1 and 2. Whether both steps are enzymatic in nature is unknown at the present time. However, the postulated intermediate 2-iminofructose-6-PQ is very probably unstable since it has never been synthesized. Therefore, the deamination per se could occur non-enzymatically. Experiments are now in progress to investigate this point. SUMM~Y k 150-fold purification of an enzyme system provisionally called "phosphoglucosaminisomerase" has been obtained from Escherichia coli lyophilized cells. This preparation converts 95-100% of the added glucosamine-6-PO4 to stoichiometric amounts of ammonia and ketose. Enzymatic and paper chromatographic analysis of the reaction products identified the ketose as fructose-6-PO~. The enzyme is active over a broad pH range with maximum ketose formation occurring at pH 7.8. Evidence is presented which indicates that the enzyme requires a sulfhydryl group for its activity. REFERENCES
1. WOLFE,J. B., MORITA,R. Y., AND NAKADA,I-I. I., Arch. Biochem. and Biophys. 64, 480 (1956). 2. 3. 4. 5. 6. 7. 8. 9.
WOLFE, J. B., AND NAKADA,H. I., Arch. Biochem. and Biophys. 64, 489 (1956). SOODAK,M., Bacteriol. Proc. 1955, 131. KORNBERG, A., J. Biol. Chem. 182, 805 (1950). PARTRIDGE, S. M., Biochem. J. 42, 238 (1948). BERSlN, T., AND MfrLLER, A., Helv. Chim. Acta 35, 475 (1952). ~V~UKHERJEE,S., AND SRIVASTAVA,H. C., Nature 169, 330 (1952). HANES, C. S., AND ISHERWOOD,F. A., Nature 164, 1107 (1949). BARRON,E. S. G., AND SINGER, T. P., J. Biol. Chem. 157, 221 (1945).
66, 333-339 (1957)
Glucosamine Degradation by Escherichia Coll. III. Isolation and Studies of "Phosphoglucosaminisomerase"~ Jack B. Wolfe 2, Barbara B. Britton and Henry I. Nakada From the Scripps Clinic and Research Foundation, La Jolla, California Received May 28, 1956 INTRODUCTION
Previous work in this and other laboratories have demonstrated that a n e n z y m e s y s t e m f r o m Escherichia coli is c a p a b l e of d e a m i n a t i n g gluc o s a m i n e - 6 - P Q (1-3). F u r t h e r , evidence i n d i c a t i n g a n isomeric conv e r s i o n of glucosamine-6-PO4 to f r u c t o s e - 6 - P Q a n d a m m o n i a h a s been p r e s e n t e d (2). I n these earlier r e p o r t s , however, t h e n a t u r e of t h e enz y m e s y s t e m concerned w i t h this c o n v e r s i o n was n o t e l u c i d a t e d . T h e p r e s e n t c o m m u n i c a t i o n p r e s e n t s d a t a on t h e i s o l a t i o n a n d f u r t h e r p u r i f i c a t i o n of t h e e n z y m e s y s t e m " p h o s p h o g l u c o s a m i n i s o m e r a s e " a n d s t u d i e s on some of its p r o p e r t i e s . METHODS
Determination of glueose6-PO4 was carried out by the use of Zwischenferment obtained from Armour and Company and further purified by acid precipitation at p g 4.4 as described by Kornberg (4). The final product was free of phosphoglucoisomerase as indicated by its failure to form ketose from glucose-6-PO4 under the experimental conditions employed. Ascending paper chromatographic analysis was carried out using conventional procedures. The synthesis of glucosamine6-PO4 and the method for determination of ammonia and ketose have been described previously (2). The reported values in all cases are corrected for substrate and enzyme controls incubated independently during the course of the reaction. The experimental conditions are reported in the legends of the text. 1 This investigation was supported in part by Research Grant #291 (C2) from the Division of Research Grants and Fellowships of the National Institutes of Health, U. S. Public Health Service. Charles Willard Stimson Postdoctoral Fellow in Biochemistry. Present address: Department of Medical Microbiology, University of Southern California, Los Angeles, California. 333
334
J . B . W O L F E ~ B . B . B R I T T O I ~ A N D H . I . NAKKDA_
TABLE I Phosphoglucoisomerase and " Phosphoglucosaminisomerase" Activities before and after Ca~(P04)2 Gel Treatment The reaction mixture contained: 0.5 ml. enzyme, 4.4 umoles glucosamine-6PO4, and 0.01M phosphate buffer pH 7.5 to a total volume of 1.0 ml. Incubation 24°, air atmosphere. Preparation
First purification b Supernatant after addition ~ ml, Ca3(PO~)2 (14.5 mg. dry wt,) Supernatant after second addition of ~ ml. Ca3(PO4)2 Supernatant from third addition of Ca3(PO~)2 (~ ml.) Elution of third Ca3(P0,)2 adsorption with pit 7.0 P0, buffer (0.01 M)
Specific activitya of Glucosamine Glucose-5-PO~ 5-P04
142 189.5
151 162.5
205 24.1 2390
54.3 0 0
a Micrograms of ketose/min./mg, protein N. b Partially purified preparation [see Ref. (2)]. Purification of "Phosphoglucosaminisomerase" Glucosamine-grown lyophilized cells of Escherichia coli were extracted and partially purified as previously described (2).3 T o a given volume of the partially purified extract (pH 6.0) containing 11.9 rag. protein in 20 ml. was added small increments of calcium phosphate gel (dry weight 29 mg./ml.). The gel was removed b y centrifugation, and the supernatant fluid was tested for its ability to convert glucosamine-6-PQ or glucose-6-PO4 to ketose. Table I presents typical data obtained b y this procedure. I£ can be seen that phosphoglucoisomerase was more readily adsorbed b y the calcium phosphate gel than "phosphoglucosaminisomerase." Elution 'of the third calcium phosphate gel precipitate with 10 ml. of 0.01 M phosphate buffer (pH 7.0) resulted in a highly purified phosphoglucosaminisomerase preparation that was free of phosphoglucoisomerase activity. As shown in Table I, the partially purified preparation, after fractional calcium phosphate gel adsorption and elution, was further purified 17-fold. This preparation does not form ketose from acetylglucosamine-6-PO~. The preparation which represented an approx3 In contrast to glucose-grown cells, the specific activity of the partially purified preparation toward glucosamine 6-phosphate and glucose 6-phosphate were 142 and 151, respectively. These values represent a 17-fold increase in deaminating activity and an 18-fold decrease in phosphoglucoisomerase activity when compared with the partially purified preparation obtained from glucose-grown cells.
GLUCOSAMINE DEGRADATION. III
335
TABLE II Relationship of Fructose Formation to Ammonia Production during Glucosamine-6-P04 Deamination
The reaction mixture contained: enzyme (11.1 ~g. prot. N.), 11.3 ~moles glucosamine-6-PO4,0.01 M phosphate buffer, pit 7.5, to a total volume of 2.7 ml. Incubation 24°, air atmosphere. Time, rain.
Exp~i~ent Productdetermined 1 Ammonia, ~moles Fructose, ~moles 2 Ammonia, ~moles Fructose, t,moles
20 4.3 3.8
40 5.3 5.8
60 7.1 7.0
80 8.2 8.5
3.8
5.4
7.0
8.3
3.5
5.3
7.9
8.7
120 10.1 10.5 9.9 10.3
200 10.9 11.2 10.7 11.0
imately 150-fold purification of the glueosamine-6-PO4 deamination system was the one used for further studies. Table I I gives ketose and ammonia values obtained during the degradation of 11.3 ~moles of glucosamine-6-PO4 b y the highly purified preparation. The results indicate that approximately equivalent amounts of ammonia and fruetose-6-PO~ are produced during the deamination of glueosamine-6-PO4. These results conclusively show that a ketose is the initial product of glucosamine-6-PO4 deamination. Approximately 95-100 % of the added ghicosamine-6-P04 can be accounted for as ketose at the completion of the reaction (Table II). This would indicate that the reaction is far to the right and, in a physiological sense, is probably irreversible. The effect of p H on ketose formation from glucosamine-6-P04 was determined. The results given in Table I I I indicate that the highly purified "phosphoglucosaminisomerase" was active over a broad p H range with a maximum of ketose formation at approximately 7.8. These results a r e quite different from that reported with the partially purified preparation obtained from glucose-grown cells (2). One possible explanation for the variance in p H optima for ketose formation between preparations could be due to a greater acid sensitivity of the highly active phosphoghicoisomerase present in the partially purified preparation. Thus, in the presence of glucosamine-6-PO4, which inhibits phosphoglucoisomerase, an enhanced inhibition of fruetose-6-PO4 removal to glucose-6-P04 might occur under acid conditions. Product Analysis
From our present knowledge of the enzyme system responsible for the deamiaation of glucosamine-6-PO4, the products appear to be ammonia and a ketose
336
J. B. WOLFE~ B. B. B R I T T O N AND H. I. N A I ~ D A
TABLE I I I
Effect of pH on Rate of Deamination of Glucosamine-6-P04 by Purified "Phosphoglucosaminisomerase" Reaction mixture contained: 0.25 ml. of enzyme (2.4 t~g. prof. N.), 4.4 #moles glucosamine-6-PO4,0.01 M phosphate buffer at the indicated ptt in a total volume of 1.0 ml. Incubation 24 °, air atmosphere. The reported pH values were determined at the end of the experimental period. pH
Fruct0se-6-P04 lS rain. 30 rain. ~mole ttmole
5.3 6.1 7.1 7.5 7.8 8.3 8.8
0.164 0.233 0.278 0.298 0.321 0.300 0.190
0.239 0.389 0.57 0.63 0.68 0.60 0.385
which is probably fructose-6-PO4 (2, 3). Evidence that the ketose formed is fructose-6-PO~ is summarized below. The reaction products obtained at the completion of the experiment shown in Table II were used for analysis. Employing the Elson and Morgan colerimetric method, only trace amounts of glueosamine could be detected. An aliquot of this reaction product containing 1.5 t~meles of ketose was tested for the presence of glucose-6-PO4 by the addition of triphosphopyridine nucleotide (TPN) and Zwisehenferment (which catalyzed the oxidation of glucose-6-PO4 to 6-phosphoglueonate with T P N as the cofactor). As shown in Table IV, only a negligible amount of glucose-6-PO4 was detectable. However, the addition of purified phosphoglueoisomerase (which catalyzes the interconversion of glucose6-PO4 and fructose-6-PO4) to this system converted the ketose to glucose-6-PO4 , which was then oxidized by the TPN-Zwischenferment system. Thus, the product appears to be fructose-6-PO4. TABLE IV
Product Analysis: Enzymatic Evidence for Fructose-6-P04 Reaction mixture contained: 0.5 ml. of 0.04 M glycylglycine buffer pI-I 7.5, 20 amoles MgC12,1.2 ~moles triphosphopyridine nucleotide, 0.1 rag. purified Zwischenferment, and an aliquot of the reaction mixture containing 1.5 ~moles ketose in a total volume of 2.7 ml. After the initial glucose-6-PO~ content was determined, 1 rag. of phosphoglucoisomerase was added for fructose-6-PO4 determinations. Treatment
Ketose ~moles
Glucose-6-PO4 ,u~oles
Untreated Zwischenferment Phosphoglucoisomerase -~ Zwischenferment
1.5 1.5 0.14
0.05 1.16
337
GLUCOSAiVIINE DEGR&DATIONo III
TABLE V
Product Analysis: Paper Chromatographic Evidence for Fructose-6-P04 RI
Substrate
Rf ButPhenol HAcH20
Color obtained with various reagents =~-Anisidlne
Ninhydrin
Molybdate
Reaction mixture Fructose-6-PO4 Glucose-6-PO~ Glucosamine-6-P04
0.13 0.13 0.17 0.14
0.08 0.08 0.11 0.12
Lemon-yellow Lemon-yellow Brown Brown
No color No color No color Blue
Blue
Reaction mixture after treatment with phosphatase Fructose Glucose Glucosamine. HC1
O.55 0.25
Lemon-yellow
No color
No color
0.55 0.25 0.41 0.20 0.2310.16
Lemon-yellow Brown Light brown
No color No color Blue
No color No color No color
i
Blue Blue Blue
Identification of the product as fructose-6-PO4 was obtained by paper chromatographic studies (Table V). Using the unidimensional ascending method, the original reaction mixture was chromatographed, using phenol (5) or butanolacetic acid-water (6). In every instance, only a single spot was detectable. Since the Ri values of glucosc-6-PO4, fructose-6-P04, and glucosamine-6-PO4 are relatively close together, further identification was accomplished by different color tests. The unknown spot was lemon-yellow by p-anisidine reagent (7), as was fructose-6-PQ, whereas glucose-6-PO4 gave a brown color. Further, treatment with Hanes-Isherwood phosphate reagent (8) gave a single blue spot that corresponded to the yellow fructose-6-PO4 spot given by the p-anisidine reagent. A separate ehromatogram was also sprayed with ninhydrin reagent. Known glucosaminc-6-PO4 in amounts of less than 1 ~g. was detectable by ninhydrin, but no ninhydrin-reactive spots could be shown on a chromatogram of the reaction mixture. Further identification of the ffuctose-6-P04 was accomplished by treating an aliquot of the reaction mixture with intestinal phosphatase (which was tested and found to be free of phosphoglucoisomerase activity). Chromatography of the phosphatase-treated material gave a single spot whose RI value was identical with fructose and whose color development with p-anisidine gave a yellow color identical with that of known fructose. These data indicate that fructose-6-PO4 is a primary reaction product of phosphoglucosaminisomerase.
Evidence for the Participation of a Sulfhydryl Group Initial experiments with p-ehloromercuribenzoate indicated that a concentration of 10-3 M completely inhibited deamination of glucosamine-6-PO4 by "phosphoglucosaminisomerase." This would indicate
338
ft. B. WOLFE~ B. B. BRITTON AND H. I. NAKADA
.°-I ~),~
/
Glutothione
,/
I~.--o'-""?
30
!
f .~o
o~
,
I
.
60 90 MINUTES F~G. 1. Inhibition of glucosamine-6-PO4 deamination by p-chloromercuribenzoate. Reaction mixture contained: Enzyme (1.4 ~g. prof. N.), 4.4 ~moles glucosamine6-PO4,0.01 M phosphate buffer pH 7.5, and, where indicated, 10-4 M p-chloromercuribenzoate in a total volume of 0.75 ml. At the time indicated, 0.1 ml. of 0.1 M reduced glutathione was added. Incubation 24°, air atmosphere. )<--no inhibitor. O--p-chloromercuribenzoate added.
that a sulfhydryl group was involved in the reaction (9). To substantiate this finding further, glueosamine-6-PO4 was incubated with "phosphoglucosaminisomerase" in the presence and absence of 10-~ M pehloromercuribenzoate. After 45 rain. incubation, 10-2 M glutathione was added to both reaction vessels. The results given in Fig. 1 indicate that prior to the addition of 10-3 M glutathione, the rate of glucosamine6-PO~ deamination was drastically inhibited by the presence of pchloromercuribenzoate. The addition of 10 ~moles glutathione, however, reversed the inhibition, for an increase in rate of deamination was then observed. These results indicate that sulfhydryl groups are involved in the deamination of glucosamine-6-PO4 by these preparations. The data presented clearly demonstrate that fructose-6-PO4 and ammonia are the products of an enzymatic isomerization of glucosalnine6-PO~ by an enzyme system provisionally called "phosphoglucosaminisomerase." From chemical considerations, there appear to be two steps involved in the reaction: (a) isomerization to a compound resembling 2-iminofructose-6-PO4 i and (b) a hydrolytic deamination of this postulated intermediate to form fructose-6-PO4 and ammonia. In this respect the mechanism of deamination would appear to be dependent upon an
GLUCOSAMIRTE DEGRADATION'. III
339
intramolecular oxidation and reduction taking place between carbon atoms 1 and 2. Whether both steps are enzymatic in nature is unknown at the present time. However, the postulated intermediate 2-iminofructose-6-PQ is very probably unstable since it has never been synthesized. Therefore, the deamination per se could occur non-enzymatically. Experiments are now in progress to investigate this point. SUMM~Y k 150-fold purification of an enzyme system provisionally called "phosphoglucosaminisomerase" has been obtained from Escherichia coli lyophilized cells. This preparation converts 95-100% of the added glucosamine-6-PO4 to stoichiometric amounts of ammonia and ketose. Enzymatic and paper chromatographic analysis of the reaction products identified the ketose as fructose-6-PO~. The enzyme is active over a broad pH range with maximum ketose formation occurring at pH 7.8. Evidence is presented which indicates that the enzyme requires a sulfhydryl group for its activity. REFERENCES
1. WOLFE,J. B., MORITA,R. Y., AND NAKADA,I-I. I., Arch. Biochem. and Biophys. 64, 480 (1956). 2. 3. 4. 5. 6. 7. 8. 9.
WOLFE, J. B., AND NAKADA,H. I., Arch. Biochem. and Biophys. 64, 489 (1956). SOODAK,M., Bacteriol. Proc. 1955, 131. KORNBERG, A., J. Biol. Chem. 182, 805 (1950). PARTRIDGE, S. M., Biochem. J. 42, 238 (1948). BERSlN, T., AND MfrLLER, A., Helv. Chim. Acta 35, 475 (1952). ~V~UKHERJEE,S., AND SRIVASTAVA,H. C., Nature 169, 330 (1952). HANES, C. S., AND ISHERWOOD,F. A., Nature 164, 1107 (1949). BARRON,E. S. G., AND SINGER, T. P., J. Biol. Chem. 157, 221 (1945).