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ocH E MI sT R Y
WITHERS, SYKES, MADSEN, AND KASVINSKY
Identical Structural Changes Induced in Glycogen Phosphorylase by Two Nonexclusive Allosteric Inhibitors? Stephen G. Withers, Brian D. Sykes, Neil B. Madsen,* and Peter J. Kasvinskyl
ABSTRACT: Recent kinetic studies on the regulation of glycogen phosphorylase by the synergistic inhibitors caffeine and glucose, in conjunction with X-ray crystallographic data, have led to the proposal of a molecular mechanism for this process in which the two effectors bind at separate sites. These effects have now been studied in solution at two different structural levels by other techniques. Ultracentrifugation studies of the quaternary structure of phosphorylase a showed the formation of dimers from tetramers on addition of these ligands independently and cooperative promotion of dimer formation when added together. The effect of the ligands on tertiary structure was investigated by studying the protection against iodoacetamide inactivation of enzyme activity afforded by these ligands. Phosphorus-3 1 ( 3iP) nuclear magnetic resonance studies of
It
is well-known that glucose acts as an allosteric inhibitor of both giycogen phosphorylase a and b (EC 2.4.1.1) (Wang et al., 1965; Wang & Black, 1968; Helmreich et al., 1967; Graves &k Wang, 1972). The nalure of this inhibition was clarified on the X-ray crystallographic identification of the binding site of glucose as the active site of the enzyme (Sygusch et al., i 977). Further crystallographic studies (Kasvinsky et al., 1978a) identified a negative effector site which binds purine nucleosides and bases in a crevice on the exterior of the molecule, I O A from the active site. This site is presumably the one originally suggested by calorimetric studies as a secondary binding site for A M P (Ho & Wang, 1973). It was later shown to bind adenine or adenosine more tightly (Morange et al., 1976). A combination of kinetic and crystallographic studies demonstrated that compounds binding at this site show competitive inhibition with respect to glucose 1-phosphate at the active site, yet by definition the inhibition is allosteric (Kasvinsky et al., 1978a,b). Studies with caffeine, a strong inhibitor, also showed a synergistic relationship with the inhibition by glucose binding a t the active site. A similar kinetic effect could therefore be observed on binding of dissimilar ligands at distinct sites, as well as cooperative interactions between them. Similar results have been obtained with phosphorylase 6.‘ It therefore seemed of interest to investigate by solution techniques the nature of the molecular changes induced in the molecule by these ligands at both the tertiary and quaternary levels. Comparison of results obtained in this way with structural information obtained by X-ray crystallographic techniques should yield a greater insight into the molecular basis for this control mechanism. The present paper describes such
the catalytically essential pyridoxal phosphate residue of adenosine 5’-phosphate (AMP) activated phosphorylase b indicated a conversion from the active to the inactive form, possibly involving a protonation of the coenzyme phosphate, on addition of either of these ligands. This suggests a similar active-site environment in the two cases, identical with that observed in the absence of the activator, AMP. The effect of these ligands on the 31Presonance of the analogous adenosine 5’-0-thiophosphate activator was also similar. Despite separate and distant binding sites for the two inhibitors, the effects on the gross structure of the enzyme at the quaternary and tertiary levels and their effect on a catalytically essential group are both identical and cooperative.
a study using several different physical and chemical techniques. An ultracentrifugal analysis was employed to investigate changes in quaternary structure. Some previous work of this kind has been carried out by using other ligands (Wang et al., 1965; Metzger et al., 1967). The differential inactivation of the enzyme upon iodoacetamide modification of sulfhydryl groups in the presence and absence of these ligands was used as an indicator of enzymic conformation. This approach has been used previously with phosphorylase to study changes induced by substrates and activators (Madsen et a]., 1973, 1976). Information on the active site was obtained by observation of the phosphorus-3 1 nuclear magnetic resonance signal of the catalytically essential pyridoxal phosphate residue. Previous studies (Feldmann & Hull, 1977) have demonstrated the sensitivity of this signal to activation of the enzyme but not to its inhibition. Supporting evidence from other kinetic and X-ray crystallographic studies is also presented.2 Materials and Methods Caffeine, icdoacetamide, and buffer chemicals were obtained from Sigma Chemical Co., except for DTT3 which was obtained from Bio-Rad Laboratories. AMPS was obtained from Boehringer-Mannheim, glucose was from Fisher Chemical Co., and D 2 0 was from Bio-Rad Laboratories. A Radiometer P H M 62 p H meter was used for all p H measurements, and those measurements made in D 2 0 buffer are uncorrected. Rabbit muscle phosphorylase b was prepared by the method of Fischer & Krebs (1 962) using mercaptoethanol instead of Kasvinsky et al. (unpublished experiments). After this paper was submitted, a report appeared by Hoerl et al. (1979) which showed that glucose has effects on the 3’P N M R spectra of pyridoxal 5’-deoxymethylenephosphonate reconstituted phosphorylase a which are similar to those we describe herein for phosphorylase b. Abbreviations used: DTT, dithiothreitol; AMPS, adenosine 5’-0thiophosphate; N M R , iiuclear magnetic resonance; Mops, 3 - ( N morpho1ino)propanesulfonic acid; PLP, pyridoxal phosphate; Bes, N,Nbis(2-hydroxyethyl)-2-aminoethanesulfonic acid; glucose- 1-P, a-D-glucopyranosyl 1-phosphate. I
‘From the Department of Biochemistry and the M R C Group on Protein Structure and Function, University of Alberta, Edmonton, Albeita, T6G 2H7 Canada. Receiced June 15, 1979; revised manuscript received September 6, 1979. This work was supported by Grant MRC MA 1414 from the Medical Research Council of Canada and by the M R C Group on Protein Structure and Function. *Present address: Department of Biochemistry, Marshall University School of Medicine, Huntington, WV 25701.
0006-2960/79/0418-5342$01.00/0
0 1979 American Chemical Society
STRUCTURAL CHANGES IN GLYCOGEN PHOSPHORYLASE
cysteine and recrystallized at least 3 times before use. Phosphorylase a was prepared from phosphorylase b with phosphorylase kinase (EC 2.7.1.38) (Krebs et al., 1964). Protein concentration was determined from absorbance measurements a t 280 nm, using the absorbance index, El,,", of 13.2 (BUC & BUC, 1968). Rabbit liver glycogen (type 111) purchased from Sigma Chemical Co. was purified on a Dowex I-CI column and assayed by the method of Dishe (Ashwell, 1957). The concentration of glycogen is expressed as the molar equivalent of its glucose residues. lodoacetamide was recrystallized according to Battel et al. (1968). Ultracentrifugal experiments were performed on a Spinco Model E analytical ultracentrifuge at a rotor speed of 56000 rpm and a temperature of 20 i 1 "C. Sedimentation coefficients determined from Schlieren patterns were corrected for the viscosity and density of the buffer to water at 20 'C. Sedimentation experiments were carried out a t pH 7.0 in IO mM Bes, 1 mM EDTA, and 2 mM DTT. Protein concentration was 5 mg/mL. Studies of the effects of caffeine and glucose on the rate of iodoacetamide inactivation of phosphorylase a were carried out by incubating enzyme (5 mg/mL) in IO mM Bes buffer containing 0.15 mM mercaptoethanol and IO mM EDTA (pH 6.8), 30 "C, with IO mM recrystallized iodoacetamide in the presence or absence of caffeine or glucose. Samples were withdrawn a t timed intervals, diluted with 20 mM glycerophosphate, 1.0 mM EDTA, and 20 mM mercaptoethanol (pH 6.8),and assayed. Initial reaction rates were determined by the Fiske-Subbarow phosphate analysis in the direction of saccharide synthesis, as described by Engers et al. (1970). Reaction mixtures were 0.5 mL and contained 2 mM sodium @-glycerophosphate(pH 6.8), 0.15 mM EDTA, 1 mM DTT, 28 m M glycogen, 75 mM glucose-I-P, and 2-4 pg of phosphorylase a. "P N M R spectra were recorded a t 109.29 M H z on a Bruker HX270 superconducting spectrometer operating in the Fourier transform mode with quadrature phase detection at 28 "C. Exponential line broadening used prior to Fourier transformation was generally 20 Hz, and all line width data have been corrected for this. A spectral width of i5000 Hz was generally employed, with a 50-70' pulse angle (15-20 ps) and a repetition time of 2.0 s. Sample size was 1.5 mL in a IO-mm tube, with enzyme concentration around I mM in monomers. The buffer used in most of the N M R experiments was 50 mM triethanolamine hydrochloride, 100 mM KCI, 1 mM DTT, and 1 mM EDTA (pH 6.8 meter reading) made up in D 2 0 . Mops buffer, used in one N M R experiment, contained 100 mM 3-(Nmorpho1ino)propanesulfonic acid, 50 m M mercaptoethanol, and 2 m M EDTA (pH 6.8) made up in D,O. The D 2 0 present in the buffer was used for fieldJfrequency lock, and a 1.0-mm tube containing 85% phosphoric acid was inserted for chemical shift referencing. A M P was removed from phosphorylase b used in N M R experiments by two extended dialyses against 500 volumes of the triethanolamine buffer made up with H,O containing 50 m M glucose, followed by dialysis against 50 volumes of this same buffer containing 50 mM glucose and charcoal (1 g) in suspension. Further dialysis against H 2 0 N M R buffer and finally against smaller volumes of D,O N M R buffer removed glucose and introduced the D,O. Enzyme prepared in this way gave no observable signal for A M P in the N M R spectrum. Solutions of effectors dissolved in D,O N M R buffer at pH 6.8 were added directly to the N M R tube as required,
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FIGURE 1: Effect of inhibitory lipands on the sedimentation properties of phosphorylase a. (a) (top) 1 mM AMP, 13.3 S; (bottom) control, 13.5 S; (b) (top) 0.05 m M caffeine, 12.7 S; (bottom) 5 mM glucose, 12.6 S; (c) (top) I O mM glucose + 0.05 mM caffeine, 8.9 and 13.1 S: (bottom) 5 mM glucose + 0.05 mM caffeine. 8.9 and 12.5 S; (d) (top) 2 mM caffeine,8.7 S (bottom) 50 mM glucose, 8.5 S. Pictures were taken 24 min after attaining full speed. Conditions were as explained under Materials and Methods.
A BASIC program written for a NOVA 1220 computer was used to perform a complete line shape analysis of the binding of AMPS. This program calculates the complete line shape according to the modified Bloch equations [see, for example, Pople et al. (1959)], assuming a two-site exchange situation. The observed line shape is influenced by the natural line width, as measured in the absence of exchange, and a line broadening contribution from exchange of this species between two sites, Le., free and bound AMPS. Introduction of estimated values for the natural line widths Avo and chemical shifts 6 of the free and bound AMPS plus estimated values for the off-rate k-, and dissociation constant KO for AMPS binding allowed simulation of a theoretical spectrum which could be fitted to the observed spectrum by adjustment of the parameters entered. The use of this approach requires the assumption of direct proportionality between peak area and concentration of the respective species. The fast exchange condition for the spin-lattice relaxation rates, T I T l B
