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Ad-Glucose As A Proc. Nati Acad. Sci. USA Vol. 79, pp. 3716-3719; June 1982 Biochemistry Catalytic mechanism of glycogen phosphorylase: Pyridoxal(5')diphospho(1)-a-D-glucose as a transition-state analogue (pyridoxal 5'-phosphate/pyridoxal 5'-diphosphate/glucosyl, transfer/phosphate-phosphate interaction) MITSURU TAKAGI, TOSHIO FUKUI*, AND SHOJI SHIMOMURA Institute of Scientific and Industrial Research, Osaka University, Suita, Osaka 565, Japan Communicated by Edmond H. Fischer, March 15, 1982 ABSTRACT Pyridoxal(5')diphospho(1)-a-D-glucose was used pearance of this spectrum and its replacement with one char- to reconstitute glycogen phosphorylase b (1,4-a-D-glucan:ortho- acteristic of pyridoxal, 5'-diphosphate (PLPP) bound. to the en- phosphate a-D-glucosyltransferase, EC 2.4.1.1) from rabbit mus- zyme. Therefore, glucose was released from the synthetic cle, replacing the natural pyridoxal 5'-phosphate coenzyme. In- coenzyme. cubation ofthe reconstituted enzyme alone resulted in the gradual This paper describes the results of the identification of pyr- cleavage ofthe synthetic cofactor to pyridoxal 5'-phosphate, which idoxal compounds produced from the enzyme-bound PLPP-a- caused slow reactivation of the enzyme. The addition of malto- Glc undervariousconditions as well as the fate ofthe pentaose or glycogen altered the mode of cleavage; the cofactor radioactive was rapidly decomposed to pyridoxal 5'-diphosphate. The radio- glucose released from the PLPP-a-['4C]Glc in the presence of active glucose moiety released from pyridoxal(5')diphospho(1)-a- glycogen. The glucose was recovered in the outer chain of gly- D-['4C]glucose was incorporated into the outer chain ofglycogen, cogen, bou'nd through an a-1,4-glucosidic linkage. The glucosyl forming an a-1,4-glucosidic linkage. These results show that the transfer reaction discovered mimics the normal catalysis ofthis glucosyl transfer reaction discovered mimics the normal catalysis enzyme, supporting strongly the catalytic mechanism based on ofthis enzyme, and they strongly support the catalytic mechanism the direct phosphate-phosphate interaction between PLP and in which the coenzyme phosphate acts as. a catalyst by direct in- the substrate. teraction. with the phosphate of the substrate, forming the pyro- phosphate-like transition intermediate. MATERIALS AND METHODS Maltose, isomaltose, and oyster glycogen were purchased from The catalytic mechanism of glycogen phosphorylase (1,4-a-D- Nakarai Chemicals (Kyoto, Japan), and maltopentaose was from glucan:orthophosphate a-D.glucosyltransferase, EC 2.4.1.1) Seishin Pharmaceutical (Tokyo, Japan). Kojibiose (2-O-a-D-glu- remains unclear despite intensive investigations. However, evi- copyranosyl-D-glucose) and.nigerose (3-O-a-D-glucopyranosyl- dence has been accumulating supporting the catalytic function D-glucose) octaacetate were kindly donated by Kazuo Matsuda of pyridoxal 5'-phosphate (PLP) bound to this enzyme: (i) The (Tohoku University, Sendai, Japan). Nigerose was obtained by amino acid sequences around the cofactor-linked residues are deacetylation of the octaacetyl derivative with sodium methyl- highly conserved in phosphorylases from many sources (1-4). ate. PLPP was synthesized as reported (5). Resolution and re- (ii) There is a large enough space to allow the binding ofa bulky constitution ofrabbit muscle phosphorylase b with PLPP-a-Glc group adjacent to the phosphate group of the cofactor, which were carried out essentially as described (11). Radioactivitywas interacts with positive charges (5, 6). (iii) All the substrates bind measured in a Beckman LS-9000 liquid scintillation system. near the cofactor, and the phosphate groups of a-D-glucose 1- Synthesis of PLPP-a-Glc. PLP monohydrate (0.53 g, 2 phosphate (Glc-1-P) and PLP are located close together (7-9). mmol) was dissolved in 20 ml ofchloroform containing 0.28 ml. The phosphate group ofP11P is likely to be involved in the cat- oftriethylamine. After evaporation,, the residue was redissolved alytic mechanism. in 15 ml of chloroform containing 0.4 ml of triethylamine. Di- On the basis of the results of 31P NMR studies, Withers et phenyl phosphochloridate (0.52 ml, 2.5 mmol) was added with aL (10) suggested that the phosphorus ofthe constrained dianion stirring, and then the solution was kept at room temperature of the- coenzyme could be an electrophile which, by direct in- for 3 hrunder anhydrous conditions. The yellow syrup obtained teraction with the phosphate group of Glc-1-P, would facilitate after evaporation was shaken with. dry diethyl ether to remove the breakage ofthe glucosidic linkage. This hypothesis was sub- the excess diphenyl phosphochloridate, and then the ether was stantiated by the recent investigation carried out collaboratively removed by decantation and the residue was dried under re- by the Edmonton group and ourselves (11). Rabbit muscle phos- duced pressure. To this syrup was added tri-l-butylammonium phorylase b reconstituted with pyridoxal(5')diphospho(1)-a-D- Glc-1-P (6 mmol) in 10 ml of dry pyridine. After being left glucose (PLPP-a-Glc) demonstrated no enzyme activity but was overnight at room temperature, the solution was evaporated to slowly reactivated on prolonged incubation. The reactivation remove the pyridine. The residue was dissolved in 20 ml of was markedly diminished in the presence of maltopentaose or water, and the solution was. extracted with 20 ml ofchloroform glycogen. The 31P NMR spectrum ofPLPP-a-Glc bound to the and then with 20 ml ofdiethyl ether. The aqueous layer diluted enzyme showed that the phosphate group was constrained by with 500 ml of.waterwas applied ona Dowex 1-X8 (Cl-) column positive charges. The addition of maltopentaose, a glycogen (1.8 .x 17 cm) and eluted successively with 200 ml ofwater, 450 analogue and an alternative substrate, resulted in the disap- Abbreviations: Glc-1-P, a-D-glucose 1-phosphate; PLP, pyridoxal 5'- The publication costs ofthis article were defrayed in part by page charge phosphate; PLPP, pyridoxal 5'-diphosphate; PLPP-a-Glc, pyri- payment. This article must therefore be hereby marked "advertise- doxal(5')diphospho(l)-a-D-glucose. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 3716 Downloaded by guest on September 27, 2021 Biochemistry: Takagi et aL Proc. Natl. Acad. Sci. USA 79 (1982) 3717 ml of0.01 M HCV0.0125 M LiCi, and 500 ml of0.01 M HCl/ PLP PLPP-a-Glc PLPP 0.017 M LiCi. and absorbance Fractions (20 ml) were collected .4 4I at 390 nm was measured by taking 0.1-ml aliquots after dilution I I I I with 3 ml of 0.1 M NaOH. The compounds eluted were iden- 0.15 tified by thin-layer chromatography. Fractions containing A PLPP-a-Glc were combined and neutralized with 1 M LiOH. 0.10 The lithium salt of PLPP-a-Glc in the concentrated solution was precipitated by addition ofacetone/methanol, 4:1 (vol/vol), and converted into the sodium salt with the aid of Dowex 50W-X8 0.05 (H' form). The sodium salt of PLPP-a-Glc in the concentrated aqueous solution was precipitated with acetone and identified as described (11). 0 I PLPP-a-[14C]Glc was synthesized from PLP and ['4C]Glc-1- E 0.50 P (New England Nuclear, 294 mCi/mmol; 1 Ci = 3.7 X 1010 0Co B becquerels) according to the above procedure but in a 1/600 scale, and isolated after mixing with unlabeled PLPP-a-Glc. -cas 0.45AI avoid ~0C.) Special care was taken during the isolation to hydrolysis r 0.10 ofthe product. The-final product gave a single spot on thin-layer ed 2 U2 chromatography when detected by fluorescence and radio- as0.05 activity. 'I1 RESULTS 0 Cleavage ofPLPP-a-Glc Bound to Phosphorylase. PLPP-a- Glc bound to rabbit muscle phosphorylase b is cleaved slowly 0.10 to produce PLP and, in the presence of glycogen or maltopen- taose, more rapidly to produce PLPP. This conclusion has been drawn from the results ofreactivation and 31p NMR analysis of 0.05 the reconstituted enzyme after incubation under different con- ditions To confirm this mode of the 0 (11). cleavage, pyridoxal L I l I I I compounds formed were analyzed by Dowex 1 column chro- 10 20 30 40 matography. During incubation of the reconstitution mixture 50 60 at pH 6.6 for 3 days, the PLPP-a-Glc remained mostly un- Fraction changed, but a small amount ofPLP was formed (Fig. 1A). The FIG. 1. Analyses of the pyridoxal compounds formed from the en- amount of PLP formed corresponded to the appearance of en- zyme-bound PLPP-a-Glc. The apoenzyme (208 ,uM) was mixed with zyme activity during incubation (11). A much larger amount of 230 ,uM PLPP-a-Glc in 0.1 M sodium glycerophosphate, pH 6.5/20 mM PLP was formed from the same reconstitution mixture incu- mercaptoethanol, and the mixture was incubated at 25°C. After 20 hr, the mixture was passed through a Sephadex G-25 column (1.2 x 26 bated at pH 7.9 (Fig. 1B), in agreement with the accelerated cm) equilibrated with 0.1 M NaCl/10 mM 2-(N-morpholino)eth- reactivation at a higher pH (11). In the presence of maltopen- anesulfonic acid/10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesul- taose, PLPP-a-Glc was almost exclusively converted to PLPP fonic acid/10 mM triethanolamine/20 mM mercaptoethanol, pH 6.8, (Fig. 1C). Because PLPP does not activate apophosphorylase and eluted with the same solution. The protein solution (188 AiM) was (5), the repressed reactivation observed (11) might be due to divided into four portions, and each was mixed with 0.1 M triethanol- the small amount of PLP that had been formed before addition amine (adjusted to different pH values)/20 mM mercaptoethanol and of maltopentaose.
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