THE JOURNAL OF BIOLJJGICAL CHEMLWRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 4, Issue of February 5, pp. 2066-2090, 1990 Printed in U. 5’. A.

Identification of Lysine 15 at the Active Site in Escherichia coli Glycogen Synthase CONSERVATION OF A LYS-X-GLY-GLY SEQUENCE IN THE BACTERIAL AND MAMMALIAN ENZYMES*

(Received for publication, July 20, 1989)

Koji FurukawaS, Mitsuo Tagaya$, Masayori Inouyes, Jack Preissll, and Toshio FukuiS 1) From the $Znstitute of Scientific and Indu-strial Research, Osaka University, Zbaraki, Osaka 567, Japan, the SDepartment of Biochemistry, Robert Wood Johnson Medical School at Rutgers, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, and the llDepartment of Biochemistry, Michigan State University, East Lansing, Michigan 48824

Glycogen synthases from Escherichia coli and mam- malian muscle differ in many respects including regu- lation, sugar nucleotide specificity, and primary se- quence. To compare the structure of the active sites in these enzymes, the affinity-labeling study of the E. coli enzyme was carried out using adenosine diphosphopyr- idoxal as the reagent. The E. coli enzyme was inacti- vated in a time- and dose-dependent manner when incubated with the reagent followed by sodium boro- hydride reduction. The inactivation was markedly pro- tected by ADP-glucose and ADP, suggesting that the reagent was bound to the substrate-binding site. The stoichiometry of the bound reagent to the enzyme was approximately 1: 1. Sequence analysis of the labeled peptide isolated from a proteolytic digest of the modi- fied protein revealed that Lys” is labeled. Based on the geometry of the reagent, the t-amino group of this residue might be located close to the pyrophosphate moiety of ADP-glucose bound to the E. coli enzyme, like that of Lys3* in the rabbit muscle enzyme, which is labeled by uridine diphosphopyridoxal (Tagaya, M., Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 260, 6670-6676; Mahrenholz, A. M., Wang, Y., and Roach, P. J. (1988) J. Biol. Chem. 263, 10561-10567). The importance of the conserved sequence of Lys-X-Gly- Gly is discussed in connection with the glycine-rich region found in many nucleotide-binding proteins.

Glycogen synthase (EC 2.4.1.21) in bacteria utilizes ADP- glucose as the glucosyl donor to synthesize an ol-1,4-glucan chain in glycogen. The enzyme from Escherichia coli has been purified to homogeneity and its properties were studied (1). It shows some similarities to the catalytic properties of mam- malian glycogen synthase. However, unlike the mammalian enzyme, it is not regulated by either a covalent or noncovalent mechanism. Its sugar nucleotide specificity is also different from that of the mammalian enzyme, which uses UDP-glucose instead of ADP-glucose. The subunit molecular weight of the bacterial enzyme is about one-half that found for the mam- malian enzyme. The nucleotide sequence of the glgA gene encoding glycogen synthase in E. coli was elucidated by Kumar et al. (2). The deduced amino acid sequence shows no signifi-

* This work has been supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence should be addressed.

cant homology with that of human muscle glycogen synthase, which was also deduced from the nucleotide sequence (3). Therefore, the two glycogen synthases seem to be completely different molecular entities.

Immediately downstream from the glgA gene, there is a glgP gene encoding a-glucan phosphorylase (EC 2.4.1.1) (4, 5). The amino acid sequence deduced from the nucleotide sequence of the glgP gene (4) revealed extensive similarities over the whole molecule to those of a-glucan phosphorylases from rabbit muscle (6), yeast (7), and potato tuber (8), and of E. coli maltodextrin phosphorylase (9), even though these phosphorylases are quite different in their regulatory proper- ties and glucan specificities. The structural relationship in phosphorylases is thus quite different from that in glycogen synthases.

Tagaya et al. (10) showed that uridine diphosphopyridoxal specifically modifies a lysyl residue located at the active site in rabbit muscle glycogen synthase. Later, Mahrenholz et al. (11) identified this residue as Lys3s. It would be of interest to examine whether this structure is conserved in the bacterial enzyme or not. To solve this problem, we have modified E. coli glycogen synthase with AP2-PL1 (12, 13), a potentially reactive analogue of ADP-glucose. This paper describes the results of those experiments as well as the overproduction and purification of the glgA gene product.

EXPERIMENTAL PROCEDURES

Materials-The materials were obtained from the following sources. T, DNA ligase was from Nippon Gene. Hind111 and PstI were from Toyobo. pUBl8 was from Takara Biomedicals. Oyster glycogen obtained from Nacalai Tesque was purified as described previously (14). ADP-[%]glucose was from ICN Radiochemicals. a- Amylases from Bacillus subtilis and human saliva were from Seika- gak; Kogyo and Sigma, respectively. Staphylococcus aureus V8 pro- tease and trvosin were from Worthineton. Vvdac C4 (214TP5415) -_ and Cl8 (218TP5415) columns were from the Separation Group. Isopropyl @-D-thiogalactopyranoside was from Nacalai Tesque. APB- PL was synthesized as described previously (12).

Construction of Plasmid pEGS-Plasmid pFY140 contains the entire glgA gene (4), but its direction is opposite for expression under the control of luc promoter. To achieve high levels of the glgA expression, pFYl40 was digested with Hind111 and ligated with pUC18. The direction of the glgA gene was checked by digestion of the resulting plasmid with PstI. The plasmid in which the glgA gene is under control of lac promoter was designated PEGS.

Protein Concentration-Protein concentration was determined by the method of Bradford (15) and calculated by using a molar absorp- tion coefficient of 2.0 x lo6 M-’ which had been determined on the basis of amino acid analysis of the purified protein.

Assay of Glycogen Synthase Activity-Glycogen synthase was as-

’ The abbreviations used are: AP2-PL, adenosine diphosphopyri- doxal; HPLC, high performance liquid chromatography.

2086

by guest on March 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

E. coli Glycogen Synthase

sayed by the filter assay method as described by Thomas et al. (16). The assay medium contained 50 mM N,N-bi@hydroxyethyl)glycine (pH 8.5), 25 mM potassium acetate, 10 mM dithiothreitol, 0.5 mg/ml oyster glycogen, 0.5 mg/ml bovine serum albumin, and 0.7 mM ADP- [“Clglucose. The medium was incubated at 30 “C for 10 min. Filter papers were placed in glass scintillation vials containing about 10 ml of toluene containing 0.4% 2,5-diphenyl-oxazol and 0.01% 1,4-bis(5- phenyl-2-oxazolyl)benzene and counted with a Beckman 9000s scin- tillation system. One unit of enzyme activity was defined as the amount that catalyzes the incorporation of 1 rmol of glucose into glycogen/min under the above conditions.

Purification of Glycogen Synthase-PEGS was transformed into E. coli JM109 as described by Maniatis et al. (17). The cells were grown on 2 liters of L broth containing 50 pg/ml ampicillin for 2 h, and then isopropyl /3-D-thiogalactopyranoside was added at a final con- centration of 0.5 mM. The cells were cultured for another 6 h and collected by centrifugation. They were suspended in 50 mM glycylgly- tine buffer (pH 7.0) containing 0.2 mM dithiothreitol and disinte- grated with a French press (Otake Seisakusho type 5501-M). The cell debris collected by centrifugation was discarded, and 2 mg of bacterial n-amylase and 500 units of salivary cu-amylase were added to the supernatant to digest the glycogen. The solution was gently stirred overnight at room temperature and then centrifuged at 78,000 X g for 90 min.

To the supernatant was added solid ammonium sulfate to 25% saturation. After standing for 1 h, the precipitate formed was collected by centrifugation at 23,000 X g, dissolved in 50 ml of 50 mM Tris- HCl (pH 7.5) containing 0.2 mM dithiothreitol and 10% glycerol (buffer A), and applied to Q-Sepharose (HR 16/10) that had been equilibrated with buffer A. The proteins were eluted with a linear gradient from buffer A to the same buffer containing 0.6 M sodium chloride over 50 min with a flow rate of 5 ml/min on the fast protein liquid chromatography separation system (Pharmacia LKB Biotech- nology Inc.). Fractions containing glycogen synthase activity were pooled, and the enzyme was precipitated by adding ammonium sulfate to 70% saturation. The precipitate was collected by centrifugation at 23,000 X g for 10 min, dissolved in a minimum volume of 50 mM 2- glycerophosphate buffer (pH 7.0) containing 0.2 mM dithiothreitol and 10% glycerol, and dialyzed against the same buffer overnight. Insoluble materials were removed by centrifugation, and the super- natant was used as the purified enzyme.

Gel Electrophoresis-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 10% acrylamide was carried out according to Laemmli (18).

Stoichiometry of Inactivation-Glycogen synthase (40 pM) was incubated with various concentrations of APS-PL at 20 “C for 30 min, reduced by sodium borohydride, and then applied to a centrifugal gel filtration column (19). The residual enzyme activity and the concen- tration of the enzyme in the passed-through fraction were measured. The amount of the bound label was determined by the fluorescence derived from the label (excitation at 330 nm and emission at 395 nm) after denaturation of the enzyme with 0.5 M Tris-HCl (pH 8.5) containing 10 mM EDTA and 6 M guanidine hydrochloride.

Isolation of AP2-PL-labeled Peptides-Glycogen synthase (3.0 FM) was incubated with 50 pM APZ-PL in a total volume of 10 ml at 20 “C for 30 min. Then the protein was reduced with sodium borohydride, dialyzed against water, and finally lyophilized. The lyophilizedprotein was reduced with dithiothreitol and S-pyridylethylated (20). The S- pyridylethylated enzyme was dialyzed against water and lyophilized. The lyophilized protein was digested by trypsin with an enzyme to substrate ratio of 1:50 (w/w) at 37 “C for 24 h. The APp-PL-labeled peptides were purified on a Gilson HPLC system with the following solvents: A, 0.1% trifluoroacetic acid, and B, 0.088% trifluoroacetic acid containing 60% acetonitrile.

Sequence Study-Amino acid sequence was determined with an Applied Biosystems model 477A sequencer linked with an Applied Biosystems model 120A phenylthiohydantoin analyzer.

Amino Acid Analysis-Amino acid was analyzed on a Hitachi 835 amino acid analyzer using o-phthaldialdehyde after 6 N HCl hydrol- ysis of samples in evacuated tubes. Proline was not quantitatively determined in this system.

RESULTS

Expression and Purification of Glycogen Synthase-In the presence of isopropyl /3-D-thiogalactopyranoside, E. coli JM109 cells carrying PEGS overexpressed the protein with

an apparent molecular weight of 48,000 (Fig. 1, lane 3). This value corresponds to the subunit molecular weight of E. coli glycogen synthase (1). Nontransformed JM109 itself did not produce this protein (Fig. 1, lane 2). In accordance with a large expression of the 48-kDa protein, JM109 cells carrying PEGS showed glycogen synthase activity 240-fold higher than that of the nontransformed cells. Thus, we concluded that this protein is the product of the glgA gene.

The overexpressed protein was purified as described under “Experimental Procedures.” To achieve a good recovery of the enzyme in the ammonium sulfate fractionation, it is important that the harvested cells are suspended in the min- imum volume of buffer in the preceding step. Table I sum- marizes the purification of the enzyme, and Fig. 1 depicts the results of sodium dodecyl sulfate-polyacrylamide gel electro- phoresis in each step of the purification. The apparent in- crease in the total enzyme activity at the Q-Sepharose step is probably caused from the depressed activity at the preceding

123456

FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electro- phoresis. Lane 1, bovine serum albumin (M, 67,000), egg albumin (43,000), and carbonic anhydrase (30,000); lane 2, the crude extract from E. coli JM109 cells; lane 3, the crude extract from E. coli JM109 cells carrying PEGS; lane 4, the 78,000 x g supernatant; lane 5, ammonium sulfate precipitate; lane 6, Q-Sepharose.

TABLE I Summary of purification of E. coli glycogen synthase

Synthase was from a 2-liter culture of E. coli JM109 carrying PEGS.

Steps Protein Total Specific activity Purification

Crude extract 78,000 X g Superna-

tant Ammonium sulfate

precipitate Q-Senharose

w unit unitlmg -fold 700 6320 9.0 1 552 4390 8.0 0.9

52 2200 42.3 4.7

21 3700 177 20

by guest on March 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2088 E. coli Glycogen Synthase

step due to the contaminating amylase. The final preparation of the enzyme showed more than 90% purity (Fig. 1, lane 6). The specific enzyme activity of the final preparation is com- parable to that of the enzyme purified from E. coli B cells (1). About 20 mg of glycogen synthase was obtained from 2 liters of the cell culture.

Inactivation of Glycogen Synthase by AP2-PL-When the purified glycogen synthase was incubated with low concentra- tions of AP2-PL followed by sodium borohydride reduction, the enzyme activity was lost in a time-dependent manner, and after 30 min, it reached a plateau dependent on the reagent concentration (Fig. 2A). About 80% of the original activity was lost during incubation with 50 pM AP2-PL for 30 min. This pattern of inactivation is typical for inactivation by modification reagents containing pyridoxal phosphate (21, 22) and is probably due to an equilibrium between the Schiff base and the free aldehyde.

The inactivation was effectively protected by substrates ADP-glucose and ADP (Fig. 2B). a-D-Glucose l-phosphate and AMP offered only slight protective effects. These results suggest that AP2-PL binds to ADP-glucose- or ADP-binding sites. Fig. 3 shows the stoichiometry of inactivation. When the degree of inactivation was plotted against the molar ratio of AP,-PL to enzyme subunit, a straight line was obtained. Extrapolation of the line to 100% loss of enzyme activity gave a value of approximately 1, indicating the specific binding of 1 mol of AP2-PL to each enzyme subunit.

AR-P1 ! Enqnm

FIG. 3. Stoichiometry of the inactivation. Stoichiometry of the inactivation was measured as described under “Experimental Procedures.

0 0 10 20 31

Time lmin)

FIG. 2. Inactivation of E. coli glycogen synthase by APZ- PL. A, effect of the concentration of the reagent. The inactivation mixture (200 ~1) contained 50 mM sodium glycerophosphate (pH 7.0), 0.2 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 2 pM enzyme, and APz-PL at 5 pM (Cl), 10 KM (0), 20 FM (A), or 50 FM (0). The mixture was incubated at 20 “C for various times, and 25 ~1 was withdrawn, and mixed with 1.5 ~1 of 30 mM sodium borohydride. After dilution enzyme activity was measured. B, effect of substrates on the inacti- vation. Glycogen synthase was incubated with 50 j.tM APZ-PL in the presence of 0.5 mM ADP-glucose (U), 0.5 mM ADP (I), 0.5 mM AMP (A), glucose l-phosphate (O), or none (A). Other conditions were essentially the same as above.

J 11 J”.

0 20 40 Time lmlnl

FIG. 4. Elution profile of a V8 protease digest of the APz- PL-labeled peptide. The V8 protease digest of the AP,-PL-labeled peptide was applied to a Vydac Cl8 column. The peptides were eluted with a linear gradient of buffer (B) from 0 to 100% over 50 min. Absorbance at 214 nm and the fluorescence (excitation at 330 nm and emission at 395 nm) of the effluent were continuously monitored.

Isolation and Sequencing of the AP2-PL-labeled Peptide- To determine the site labeled by AP2-PL, the APz-PL-labeled enzyme was S-pyridylethylated and digested by trypsin, as described under “Experimental Procedures.” The digest was chromatographed on the HPLC system using a Vydac C4 column. One predominant fluorescent peak was observed, indicating the binding of the label to a specific site. Sequence analysis of this labeled peptide revealed that its amino-ter- minal sequence is the same as that of the native protein (data not shown). This peptide was further digested with V8 pro- tease and then applied to a Vydac Cl8 column. The labeled peptide was eluted again as a single peak (Fig. 4). Sequence analysis of this peptide revealed that its structure is Met-Phe- Pro-Leu-Leu-X-Thr-Gly-Gly-Leu-Ala-Asp, where X repre- sents an unidentified amino acid (Table II). This structure is consistent with the amino acid composition (Asp,Thri. GlyZAlalMet1Leu3Phe# and is identical to that from Met” to Asp” except for Lys” in the complete amino acid sequence

’ In the amino acid analysis, lysine was not detectable, and proline could not be quantitatively determined.

by guest on March 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

E. coli Glycogen Synthase 2089

TABLE II enzyme was obtained by replacing Gly” or Gly*’ by alanine Sequencing of APp-PL-labeled peptide (43). These results clearly indicate that the flexible loop is

Cycle Residue Yield important for the binding of substrates in adenylate kinase.

2 3

5 6 7 8 9

10 11 12

Met Phe Pro Leu Leu ND

Thr GUY Glr Leu Ala ASP

pd 194 111 134

41 25

ND 34 16

117 33 39 36

a Not detectable.

of E. coli glycogen synthase (2). Since an APP-PL-labeled lysyl residue cannot be positively identified (13), we concluded that Lys” is labeled by AP2-PL.

DISCUSSION

The results of the present investigation provide evidence for the presence of Lysi’ at the active site in E. coli glycogen synthase. Based on the geometry of the modified reagent, the c-amino group of Lys” is probably located close to the pyro- phosphate moiety of ADP-glucose in the E. coli enzyme, like that of Lys3’ in the rabbit muscle enzyme which is labeled by uridine diphosphopyridoxal (13, 14). Upon comparison of the amino-terminal sequences among glycogen synthases from E. coli (2), rabbit muscle (ll), and human muscle (3), we have found that a sequence of Lys-X-Gly-Gly (X represents an unspecified residue) containing the labeled lysyl residue is conserved in the three enzymes, although the bacterial and the mammalian muscle enzymes are not homologous (3). The homology seen for Lys-X-Gly-Gly in the three enzymes may be a reflection on the importance of the sequence for sugar nucleotide binding.

A motif in which several glycyl residues are close to a reactive lysyl residue has been found in many nucleotide- binding proteins. In all the glutamate and leucine dehydro- genases, a Gly-Gly-X-Lys sequence is conserved (23-31). Pisz- kiewicz et al. (32) reported that pyridoxal phosphate modifies the conserved lysyl residue in glutamate dehydrogenase. On the other hand, Lys3’ in rabbit muscle glycogen synthase was also modified by pyridoxal phosphate if a higher concentration of the reagent was used (11). Therefore, the conserved lysyl residues in glycogen synthases and dehydrogenases seem to be equivalent. Walker et al. (33) found that a Gly-X-X-X-X- Gly-Lys sequence is conserved in several ATP- and GTP- binding proteins, which include adenylate kinase (34), H+- ATPase (33), and the ras oncogene product p21(35). We have demonstrated that the conserved lysyl residues in these pro- teins are specifically labeled by adenosine or guanosine poly- phosphopyridoxals (13,36-39). Therefore, the motif of a lysyl residue in the glycine-rich region is general as the structural element of polyphosphate-binding loci.

In adenylate kinase, a segment of Gly-Gly-Pro-Gly-Ser- Gly-Lys-Gly containing the reactive Lys’i constructs a loop structure with the turn at ProI (40). Furthermore, this loop remarkably moves during the transition between two crystal forms, regarded as an induced fit of the enzyme (41). Replace- ment of Proi by glycine or valine via site-directed mutagen- esis produced the mutant enzyme having enzyme activities comparable with that of the wild-type enzyme but markedly reduced affinities for the two substrates (42). A similar mutant

We do not yet fully understand how the conserved sequence interacts with the polyphosphate moiety in glycogen synthase. One possibility is that the conserved lysyl residue interacts directly with the polyphosphate moiety of substrate sugar nucleotide, and the glycyl residues provide the flexibility in this locus, as observed in adenylate kinase. Another possibility is that the glycyl residues themselves interact directly with the polyphosphate moiety. In triose phosphate isomerase, the amide nitrogens of the 2 glycyl residues (G1yZ3* and G~Y’~~) interact with the phosphate moiety of substrate triose phos- phate (44). However, since this structure may be applicable only to monophosphate-binding sites, the former possibility is more likely. To understand the exact role of the conserved lysyl and glycyl residues in glycogen synthase, site-directed mutagenesis experiments are now in progress.

REFERENCES 1.

2.

3.

4.

5.

6.

7. 8. 9.

10.

11.

12. 13.

14. 15. 16.

17.

18. 19. 20.

21.

22. 23.

24.

25.

26.

27.

28.

29.

Fox, J., Kawaguchi, K., Greenberg, E., and Preiss, J. (1976) Biochemistry 15,849-857

Kumar, A., Larsen, C. E., and Preiss, J. (1986) J. Biol. Chem. 261,16256-16259

Browner, M. F., Nakano, K., Bang, A. G., and Fletterick, R. J. (1989) Proc. N&l. Acad. Sci. U. S. A. 86, 1443-1447

Yu, F., Jen, Y., Takeuchi, E., Inouye, M., Nakayama, H., Tagaya, M., and Fukui, T. (1988) J. Biol. C&m. 263,13706-13711

Romeo. T.. Kumar. A.. and Preiss. J. (1988) Gene (Amst.) 70. 363-376’

Titani, K., Koide, A., Hermann, J., Ericsson, L. H., Kumar, S., Wade, R. D., Walsh, K. A., Neurath, H., and Fischer, E. H. (1977) Proc. Natl. Acad. Sci. I/. S. A. 74, 4762-4766

Hwang, P. K., and Fletterick, R. J. (1986) Nature 324, SO-84 Nakano, K., and Fukui, T. (1986) J. Biol. C&m. 261,8230-8236 Palm, D., Goerl, R., Weidinger, G., Zeier, R., Fischer, B., and

Schinzel, R. (1987) 2. Naturforsch. Teil C. Biochem. Biophys. Biol. Viral. 42, 394-400

Tagaya, M., Nakano, K., and Fukui, T. (1985) J. Biol. Chem. 260,6670-6676

Mahrenholz, A. M., Wang, Y., and Roach, P. J. (1988) J. Biol. Chem. 263, 10561-10567

Tagaya, M., and Fukui, T. (1986) Biochemistry 25,2958-2964 Tagaya, M., Yagami, T., and Fukui, T. (1987) J. Biol. Chem.

262, 8257-8261 Tagaya, M., and Fukui, T. (1984) J. Biol. Chem. 259,4860-4865 Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 Thomas, J. A., Schlender, K. K., and Larner, J. (1968) Anal.

Biochem. 25, 486-499 Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular

Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Laemmli, U. K. (1970) Nature 227,680-685 Penefsky, H. S. (1977) J. Biol. Chem. 252,2891-2899 Hermodson, M. A., Ericsson, L. H., Neurath, H., and Walsh, K.

A. (1973) Biochemistry 12, 3146-3153 Shapiro, S., Enser, M., Pugh, E., and Horecker, B. L. (1968)

Arch. Biochem. Biophys. 128, 554-562 Gould, K. G., and Engel, P. C. (1980) &o&em. J. 191, 365-371 Moon, K., and Smith, E. L. (1973) J. Biol. Chem. 248, 3082-

3088 Moon, K., Piszkiewicz, D., and Smith, E. L. (1973) J. Biol. Chem.

248,3093-3107 Blumenthal, K. M., Moon, K., and Smith, E. L. (1975) J. Biol.

Chem. 250,3644-3654 Julliard, J. H., and Smith, E. L. (1979) J. BioE. Chem. 254,3427-

3438 Haberland, M. E., and Smith, E. L. (1980) J. Biol. Chem. 255,

7984-7992 McPherson, M. J., and Wootton, J. C. (1983) Nucleic Acids Res.

11,5257-5266 Valle, F., Becevil, B., Chen, E., Seeburg, P., Heyneker, H., and

Bolivar, F. (1984) Gene (Am&) 27, 193-199

by guest on March 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2090 E. coli Glycogen Synthase

30. Moye, W. S., Ammo, N., Rao, J. K. M., and Zalkin, H. (1985) J. Biol. Chem. 260,8502-8508

31. Nagata, S., Tanizawa, K., Esaki, N., Sakamoto, Y., Oshima, T., Tanaka, H., and Soda, K. (1983) Biochemistry 27,9056-9062

32. Piszkiewicz, D., Landon, M., and Smith, E. L. (1970) J. Biol. Chem. 245,2622-2626

33. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1,945-951

34. Kuby, S. A., Palmieri, R. H., Frischat, A., Fischer, A. H., Wu, L. H., Maland, L., and Manship, M. (1984) Biochemistry 23, 2393-2399

35. Yuasa, Y., Srivastava, S. K., Dunn, C. Y., Rhim, J. S., Reddy, E. P., and Aaronson, S. A. (1983) Nature 303, 775-779

36. Yagami, T., Tagaya, M., and Fukui, T. (1988) FEBS Lett. 229, 261-264

37. Noumi, T., Tagaya, M., Miki-Takeda, K., Maeda, M., Fukui, T., and Futai, M. (1987) J. Biol. Chem. 262, 7686-7692

38. Ohmi, N., Hoshino, M., Tagaya, M., Fukui, T., Kawakita, M., and Hattori, S. (1988) J. Biol. Chem. 263, 14261-14266

39. Tagaya, M., Noumi, T., Nakano, K., Futai, M., and Fukui, T. (1988) FEBS Lett. 233, 347-351

40. Dreusicke, D., Karplus, P. A., and Schulz, G. E. (1988) J. Mol. Biol. 199,359-371

41. Pai, E. F., Sachsenheimer, W., Schirmer, R. H., and Schulz, G. E. (1977) J. Mol. Biol. 114,37-45

42. Tagaya, M., Yagami, T., Noumi, T., Futai, M., Kishi, F., Naka- zawa, A., and Fukui, T. (1989) J. Biol. Chem. 264,990-994

43. Yoneya, T., Tagaya, M., Kishi, F., Nakazawa, A., and Fukui, T. (1989) J. Biochem. (Tokyo) 105, 158-160

44. Aiber, T., Banner, D. W., Bloomer, A. C., Petsko, G. A., Phillips, D., Rivers, P. S., and Wilson, I. A. (1981) Phil. Trans. R. Sot. Land. B293,159-171

by guest on March 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

K Furukawa, M Tagaya, M Inouye, J Preiss and T Fukuienzymes.

Conservation of Lys-X-Gly-Gly sequence in the bacterial and mammalian Identification of lysine 15 at the active site in Escherichia coli glycogen synthase.

1990, 265:2086-2090.J. Biol. Chem. 

  http://www.jbc.org/content/265/4/2086Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/265/4/2086.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on March 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from