Affinity Labeling of Muscle Phosphorylase b with

Transcription

1 Agric. Biol Chem., 52 (ll), , Affinity Labeling of Muscle Phosphorylase b with a-cyclodextrin-dialdehyde Shiro Takagi, Mikihiko Kobayashi,* Tadanori Urayama, Itsuko Suzawa, Kazuo Matsuda** and Eiji Ichishima Department oj Agricultural Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Miyagi 981, Japan Received April 7, 1988 Rabbit muscle phosphorylase b was modified with a substrate analog, the 2',3/-dialdehyde derivative of a-cyclodextrin (dial-a-cd). Although the inhibition of phosphorylase b by a-, /?-, and y-cyclodextrins gave rather high Ki values (10 ~25 him), the dial-cd gave much smaller Ki values of 1.2~3.5mM. Moreover, the latter inhibition was time-dependent and accelerated by higher phs and higher concentrations of dial-cd. Incorporation of the dial-cd into the enzyme was proportional to the loss ofenzyme activity and became stationary at about 1 mol of dial-cd bound to a mol of enzymesubunit. Modification was greatly suppressed by the presence of substrate glycogen. Glucose 1-phosphate was not effective. The dial-cd-modified phosphorylase b was purified by Sephadex G-75 and Con A-Sepharose column chromatography. The modified enzyme gave a single band of activity having a Kapp of 6.3% glycogen on affinity gel electrophoresis, which showed that the modified enzyme had a very low affinity for glycogen. Comparison of Kmvalues of the native and modified phosphorylase b showed that the Kmvalues for the glucan substrates increased 8 to 1 1-fold in the modified enzyme. These results suggested that the glycogen storage site of muscle phosphorylase b might be modified with dial-cd and the modified enzyme significantly decreased in the affinity for the substrate glycogen. Muscle glycogen phosphorylase [EC ] catalyzes the transfer of a glucose moiety between glucose 1-phosphate and a-l,4-glucan. Recent studies on the primary structure and X-ray crystallographic analysis of muscle phosphorylase revealed that a glycogen storage site (a-helix of residues 396 ~412) participated in the regulation of enzymeactivity.1* On the other hand, studies on the potato tuber phosphorylase showed no regulatory site in the potato enzymecorresponding to the glycogen storage site of the muscle enzyme. Instead of this site, there was a large insertion of 78 amino acids in the corresponding domain,2) and a cyclodextrin binding site adjacent to the co factor (pyridoxal 5'-phosphate) binding site was proposed.3) X-Ray studies showed that maltooligosac- To whomcorrespondence should be addressed. Present address: Iwaki-Meisei University, Fukushima 970, Japan. charides bind preferentially at the glycogen storage site, 25~30A from the pyridoxal 5'- phosphate binding site in muscle phosphorylase.4) Moreover, kinetic analysis with maltoheptaose showed that the binding affinity for the glycogen storage site was more than 20- fold higher than that for the active site.5) When one of the non-reducing ends of the highly branched glycogen molecule is captured by this site, the other one end binds readily to the active site and thus, the dimeric form of phosphorylase is stabilized and activated.1} Therefore, highly branched glycogen is a better substrate for the muscle enzymethan linear a-l,4-glucans such as amylose. However, these mechanisms are mainly based on X-ray studies, and further information on the amino acid residues that participate in the glucan

2 2710 S. Takagi et al. binding reaction must be accumulated. Periodate oxidation of the ribose ring of ATp 6-10) ADp ll) AMp 12) and UDp (uridine 5 /-diphosphate)13) gave 2',3 '-dialdehyde structures in the ribose moiety, and these dialdehyde compounds were useful affinity labeling reagents for various enzymes. In these cases, the dialdehyde group of nucleotides was supposed to give either a Schiff base6'8'13) or the dihydroxymorpholine-type structure with a specific lysine molecule in the active site.7 ll) In the case of a-cyclodextrin (a-cd), periodate oxidation with equimolar sodium metaperiodate provided 2',3'-dialdehyde structures of the average 3.5 D-glucose molecules,14) which was a potent inhibitor of various amylases and phosphorylases.15) To get information on the glycogen storage site of muscle phosphorylase, we tried affinity labeling by a dialdehyde derivative of a-cyclodextrin, which was a substrate, glycogen, analog. In this paper, we describe detailed characterization of the affinity labeling of muscle phosphorylase b with the a-cyclodextrin-dialdehyde (dial-cd). MATERIALS AND METHODS Materials. Preparation of 2',3/-dialdehyde derivative of a-cyclodextrin (dial-cd) was described in our previous paper.14) Dial-CD I, II, and II. V corresponded to the a- cyclodextrin oxidized with 1, 2, and 2.5 molar excess of sodium metaperiodate. a-glucan phosphorylase (b form) [EC ] from rabbit skeletal muscle was purified by the method of Fischer and Krebs.16) Phosphorylase a was prepared from the b form by the use of phosphorylase kinase (P-2014, Sigma Chemical Co., St. Louis, MO.) as described by Krebs.17) Inhibition ofphosphorylase with dial-cd. For the standard assay, rabbit muscle phosphorylase b (300 //g/ml) was mixed with 2.5raM dial-cd in 50mM triethanolamine buffer, ph 8.5. Incubation was conducted at 30 C in the dark. The reaction was ended by a 10-fold dilution with 40mMjS-glycerophosphate buffer (ph 6.8) at 0 C. The residual activity of phosphorylase b was assayed in the direction of glycogen synthesis as described previously.15) Effects of ph on the modification of phosphorylase were examined with 50mM HEPES buffer (2-hydroxyethylpiperazine-Af-2-ethanesulfonic acid) for ph 6.5~8.0 or 50mMtriethanolamine buffer for ph 8.5~9.5. Stoichiometric measurement. Incorporation of dial-cd into the phosphorylase b was assayed as follows: the enzyme (20mg) was modified with the dial-cd as described above. After incubation for 0 ~240min, a portion of the reaction mixture was adjusted to ph 6.8 with 1 m acetic acid at 0 C, and its activity was measured. The residual portions were put on a DEAE-Sephacel column (0.9 x 4.0cm), which was equilibrated with 40mM/?-giycerophosphate buffer, ph6.8. The column was washed with the buffer to remove the unreacted dial-cd, and then eluted with 0.35 m NaCl in the buffer to elute the enzyme. The pooled fractions of each time-point were mixed with Taka-amylase A (8.2 unite/87/ig, Seikagaku Kogyo Co., Ltd.) and incubated at 37 C for 20hr to release reducing sugars from the enzyme bound dial-cd. The reaction mixture was ultra filtered on with Molecut II GS (Millipore; exclusion range of MW10,000) and the filtered low-molecular-weight fraction was obtained. The reducing sugar derived from the enzyme bound-dial-cd was measured by a modified micro-assay method with a fluorescent reagent (BEC).18 19) The phosphorylase b was measured using an absorbancy index of A^h Isolation of modified phosphorylase. The dial-cd modified phosphorylase b was put on a Sephadex G-75 column (3.6x25cm) equilibrated with 20mM /?-glycerophosphate-1mm EDTA, ph 6.8. The void volume fraction was collected and mixed with oyster glycogen (final 0.2%), and then put on a Con A-Sepharose column (1.6 x 5.0cm) equilibrated with the above buffer containing 0.1 mm CaCl2-0.1 mm MgCl2-0.1 mm MnCl2, ph 6.8. The column was eluted with the equilibrating buffer followed by the buffer containing 20mM methyl a-dglucoside. The first peak having no affinity for the Con A- Sepharose was pooled and rechromatographed on the same column as described above. To remove a small amount of glycogen from the modified enzyme, the pooled fraction was put on a charcoal column (0.9x4.0cm), which was composed of equal volumes of charcoal and celite (No. 545). The modified enzyme, eluted with 40mM /?-glycerophosphate buffer, ph 6.8, was pooled and concentrated. The specific activity of the purified preparation of the dial-cd modified phosphorylase b was about 2 units/mg protein. Affinity gel electrophoresis. Polyacrylamide gel electrophoresis (PAGE)was done using to the gel system of Davis as described previously.20) Electrophoresis was done with 5% gel containing 0.1% glycogen at 4 C. Activity staining of phosphorylase was done by the method of Takeo and Nakamura,21' and the apparent dissociation constant (Kapp) for glycogen was determined. RESULTS Inhibition ofphosphorylase by dial-cd The potato tuber phosphorylase was eom-

3 petitively inhibited by a-, /?-, and y-cd, with Ki of0.15, 0.42, and 4.9mM, respectively.22* In contrast, the rabbit muscle phosphorylase was competitively inhibited by these CD to a much smaller extent and gave Ki values of 10~25 mm for both a and b forms (Table I). However, the inhibition of phosphorylase b by the dial-cd gave Kivalues of3.5, 1.9, and 1.2mM for a-, /?-, and y- forms, respectively (Table I). As shown in Fig. 1, the inhibition of muscle phosphorylase b by the dial-cd was greatly affected by the ph of the reaction mixture. At ph 9.5, phosphorylase b lost 75% of its activity within 20min, but about 72% of the activity was retained after the 60min incubation at ph 7.0. At ph 8.5, the k.dpp value (the apparent first-order rate constant of inactivation) was 0.018mm"1 (Fig. lb). Effects of ph on the modification of phosphorylase b were further examined by affinity gel electrophoresis with the 5% polyacrylamide gel containing CD Table I. Inhibition of Various Phosphorylases by CD Modification of Phosphorylase with Dial-CD Ki (mm) Muscle a Muscle b Potato* a-cd (3.5)b CD - 15 (1.9) 0.42 y-cd (1.2) 4.9 a Seeref. 22. b These values were the Ki value with the dial-a-cd I. 0.1% glycogen. Two different forms of the modified phosphorylase activity were observed as shown in Fig. 2. The native enzyme had a high affinity for the substrate glycogen and gave the Rm(relative mobility) value of Although the modification at ph 7.5 gave only an S component (band S with Rm0.30), ph 8.5 gaveboths andf(bandfwith Rm0.49) components, and the S component disappeared in 60min of incubation. A faint band of the activity of the F component was observed after 30min of incubation at ph 9.5. The effects of the dial-cd concentration on the inactivation of phosphorylase b were examined (Fig. 3). The kapp values were proportional to the concentration of dial-cd and gave values of the maximal rate constant, k0, of 0.303mm"1 and the dissociation constant for inactivation, A^inact, of 66.7mM (Fig. 3b). Moreover, the relationship between log [dial- CD] and log k was linear with a slope ft=0.89 (Fig. 3c). According to the definition by Levy et al.,23) a stoichiometric incorporation of the ligand gave a slope ofn= 1.0, and the present value (n=0.89) corresponded well to the stoichiometric measurement as described later. With the dial-cd of different degrees of oxidation, which were prepared by the oxidation with an equimolar (1.0 dial-cd I) to 2.5 molar excess (2.5, dial-cd II.V) of sodium metaperiodate to the a-cd, kapp values ranged from to 0.067mm"1 (data not shown). These Fig. 1. Effects of ph on the Incorporation of Dial-a-CD I into Muscle Phosphorylase b. (a): Course of inactivation with different phs. phs of the reaction mixture (50mMtriethanolamine buffer) were 7.0 (å ), 8.0 (V), 8.5 (#), 9.0 (T), and 9.5 (O). (b): Relationship between the phand the apparent rate constant of inactivation.

4 2712 S. Takagi et al. Fig. 2. Affinity Gel Electrophoresis of Muscle Phosphorylase b Modified at Different phs. phs of the reaction mixture (50 nim triethanolamine buffer at ph 7.5, 8.5, and 9.5) and the incubation times (0, 30, and 60min) are indicated at the top of gels. Arrows O, S, and F show the positions of the native phosphorylase b, S component (Rm 0.30), and F component (Rm 0.49), respectively. After the electrophoresis with 5%gels containing 0. 1 % glycogen, phosphorylase activity was detected by the iodine staining method.21) Fig. 3. Inactivation of Muscle Phosphorylase b by Different Concentrations of Dial-a-CD I. (a): Course ofinactivation with 10 (#), 20 (å ), 30 (T), 40 (V), and 50(O) him of dial-a-cd I. (b): Double reciprocal plot of the dial-a-cd I and &app. (c): Relationship between the concentration of the dial-a-cd I and log kapp values. results showed that the increase in the dialdehyde groups introduced in the CDmolecule increased the reactivity with the phosphorylase. However, to assure the specific modification ofphosphorylase, the reaction was basically conducted with the dial-cd I at ph 8.5. Characterization of the reaction Correlation between the inactivation of muscle phosphorylase b and the incorporation of dial-cd into the enzyme was quantitatively determined (Fig. 4). In this experiment, both dial-cd I and II were incubated with the enzyme and the amounts of dial-cd

5 Modification of Phosphorylase with Dial-CD Fig. 4. Stoichiometry of Inactivation of Muscle Phosphorylase b and Incorporation of Dial-a-CD. Remaining activity (%) with the dial-a-cd I (#) and dial-a-cd II (Y). Amount of the dial-a-cd I (O) and II (V) incorporated in the enzyme, which was calculated from the calibration curve with the Taka-amylase A digested dial-cd I or II, was shown by mol/mol of phosphorylase subunit. incorporated were measured after the removal of free dial-cd by column chromatography on DEAE-Sephacel. Although the dial- CD II caused more than 2-fold faster modification of the phosphorylase, an almost complete loss of activity and the stoichiometric incorporation of the dial-cd were also attained in the case of dial-cd I. Together with the result shown in Fig. 3c, we judged that the muscle phosphorylase b was specifically labeled with the dial-cd. Since the dial-cd is a structural analog of the substrate glycogen, the active site of phosphorylase might be labeled by this compound. This would be consistent with the competitive inhibition kinetics of the dial-cd for the substrate glycogen (Table I). On the other hand, recent X-ray analysis and kinetic studies showed that the muscle phosphorylase has a glycogen storage site located at the surface of the enzyme molecule.1} Binding of maltoheptaose to this site was 22-fold stronger than that to the active site.5) Therefore, the dial-cd might be incorporated into the glycogen storage site. Protection of the enzyme activity by various ligands during the modification with the dial-cd is shown in Fig. 5. Neither glucose nor glucose 1-phosphate effectively prevented the loss of enzymeactivity, but glycogen gave an obvious protection. Fig. 5. Effects of Ligands on Inactivation of Muscle Phosphorylase b by Dial-a-CD I. Phosphorylase 6 (10 /jg/ml) was modified with 2.5 mmdiala-cd I in 10mM /?-glycerophosphate, ph 6.8, in the presence of 0.5% glycogen (O) or 1mM AMP (V). Additions of 10mM glucose 1-phosphate, 10mM glucose, and 1mM caffeine gave almost the same values as the control (#). Neither an activator, AMP, nor an inhibitor, caffeine, was effective as compared with glycogen. Isolation of the modified phosphorylase Affinity electrophoresis (Fig. 2) showed that the muscle phosphorylase modified with the dial-cd retained the enzyme activity but lost the affinity for the substrate glycogen. Based on these characteristics, the modified phospho-

6 2714 S. Takagi et al. Table II. Kinetic Constants of Native and Modified Muscle Phosphorylase b Native Modified Substrate ( Ligand) Kmax Kmax K m /TT/. Km /TT/. (U/mg) (U/mg) Glycogen 0.018% % 0.06 Amylopectin 0.042% % 0.29 Glucose 10.4mM him phosphate Maltohexaose 6.1 mm mm 0.21 AMP(Xfl) 0.07mM ll mM 0.08 Fig. 6. Elution Pattern of Dial-a-CD I Modified Phosphorylase b from a Con A-Sepharose Column. The dial-a-cd I modified enzyme was eluted before the elution with 20mMmethyl a-d-glucoside shown by an arrow. The first peak (peak A) was rechromatographed. rylase was separated from the native enzyme. Gel filtration on a Sephadex G-75 column effectively removed the residual dial-cd and the resulting enzyme was mixed with 0.2% glycogen followed by affinity chromatography on a Con A-Sepharose column (Fig. 6). After the elution of a breakthrough fraction (peak A) with the buffer, the enzyme fraction (peak B) bound to the Con A via glycogen complex formation was eluted with 20mMmethyl a-dglucoside. Peak A was pooled and further purified by rechromatography on the Con A- Sepharose column. Similar to the results shown in Fig. 2, the modified enzyme from the Sephadex G-75 column gave two activity bands of the S and F components. Dissociation constants of these components were evaluated by affinity gel electrophoresis, which showed that the component S (Kapp 0.30%) had the same level of affinity for glycogen as the native enzyme (Kapp 0.21%). In contrast, the component F (Kapp 6.3%) had 30-fold lower affinity than the native phosphorylase b (data not shown). Therefore, the modification with the dial-cd clearly impaired the binding affinity of the component F for the substrate glycogen. The peak A fraction purified by the Con A- Sepharose column corresponded to the component F of the affinity gel electrophoresis. Kinetics of the modified phosphorylase The purified component F was analyzed kinetically. As shown in Table II, the Km values of modified enzyme were 0.191% and 0.315% for glycogen and amylopectin, respectively. These values were about 8 to ll-fold higher than those of the native enzyme. The Kmvalue for maltohexaose, which served as an acceptor substrate, was also higher than that of the native enzyme. In contrast, the Km value for glucose 1-phosphate (16.2mM) was rather close to that of the native enzyme (10.4mM). These results suggested that the binding of acceptor substrate was greatly impaired by the modification of muscle phosphorylase with the dial-cd. Moreover, activation of the muscle enzyme by AMPwas also affected by the modification, and the Ka value was increased about 10-fold. DISCUSSION The role of pyridoxal 5'-phosphate in the catalytic reaction of muscle phosphorylase was studied by the reconstitution of apophosphorylase with a coenzyme-substrate conjugate of pyridoxal (5')-diphospho(1)-a-D-glucose (PLPP-G).24) Kinetic analysis indicated that the imidazoyl group of His-376 acted as a nucleophile for the substrate glucose 1-phos-

7 Modification of Phosphorylase with Dial-CD 2715 phate, where PLPP-G bound to the enzyme and served as a glucosyl donor to glycogen. Other studies on the modification of muscle phosphorylase with substrate analogs were reported, where cyanogen-bromide-activated maltooligosaccharides25) or glycogen26) were covalently attached to the enzyme. In the former paper, direct evidence for the existence of the glycogen storage site was provided.25) Dialdehyde derivatives of cyclodextrins of a-, /?-, and y- forms were more potent inhibitors of muscle phosphorylase b than the native forms and had much smaller Ki values (Table I). The irreversible inhibition of enzyme was stimulated by a higher ph of the reaction mixture (Fig. 1) and a higher concentration of the dial-cd (Fig. 3a,b). Treatment of the dial-cd modified enzyme with NaBH4 gave no change in both activity and the relative mobility on affinity-page. Moreover, treatment of the modified enzyme with various reagents including cysteamine, L-cysteine ethylester,27) and Tris buffer (ph 7.5)8'13) gave no reactivation of the modified enzyme (data not shown). Therefore, the linkage between the enzyme and dial-cd would be dihydroxymorpholine-like adducts7 ll) rather than SchifT bases.6'8'13) This should be established by more precise analysis of the chemical structure of the adduct. Stoichiometric measurements between the inactivation of enzyme and the incorporation of dial-cd into the enzyme showed that the modification of enzyme with both dial-cd I and II gave a complete inactivation when about 1 mol of dial-cd was bound to 1 mol of enzyme (Fig. 4). This results is consistent with the value of the slope n=0.89 derived from the kinetic measurements (Fig. 3c), which indicates the stoichiometry of the bound dial- CDto the enzymesubunit was calculated to be 0.89mol/mol as described by Levy et al.23) Affinity-PAGE of the modified phosphorylase gave two components having different affinities for the substrate glycogen. Since the dial-cd was a substrate analog, this compound might have a high affinity for the active site and/or the glycogen storage site. However, kinetic5} and X-ray studies4* suggested that the preferential binding of maltooligosaccharides occurs at the glycogen storage site. Therefore, we tentatively suppose that the dial-cd binds to the glycogen storage site rather than the active site. Although a more definite conclusion would be provided by the amino acid sequencing of the labeled peptide from the dial-cd modified enzyme, at present, there are three pieces of evidence that support our current postulate on the modified position. First, the modified phosphorylase retained the enzyme activity as shown in Figs. 2 and 6 and Table II. If the dial-cd binds at the active site, binding of substrate glycogen would be impaired leading to the complete inactivation of the enzyme. Second, the apparent dissociation constant of the modified enzyme (component F) measured by the affmity-page method was 30-fold higher than that of the native enzyme, and the Km of modified enzyme for glycogen was 1 1-fold higher than the native enzyme. These values corresponded to the magnitude of difference in the affinity for glycogen between the active site and glycogen storage site.5) Moreover, the increase in the Kmof modified enzyme for the glucan substrates was 5 to 1 1-fold, but that for glucose 1-phosphate was only 1.6-fold (Table II). Third, protection of dial-cd modification by glucose 1-phosphate was not as effective as that by glycogen (Fig. 5). These data suggest that the protein structure of the active site may not be greatly changed by the modification with the dial-cd. Although we have no further evidence to explain the formation of component S, this species might be attributable to the combination of modified and native subunits of phosphorylase b. Because the glycogen storage site is at the very surface of the enzyme molecule, it is probable that one of the subunits of dimeric form of phosphorylase b was transitionally modified. REFERENCES 1) R. J. Fletterick and N. B. Madsen, Annu. Rev. Biochem., 49, 31 (1980).

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