©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chimeric Muscle and Brain Glycogen Phosphorylases Define Protein Domains Governing Isozyme-specific Responses to Allosteric Activation (*)

Michael M. Crerar (1)(§), Olof Karlsson (2), Robert J. Fletterick (2), Peter K. Hwang (2)(¶)

From the (1) Department of Biology, York University, North York, Ontario M3J 1P3, Canada and the (2) Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0448

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Muscle and brain glycogen phosphorylases differ in their responses to activation by phosphorylation and AMP. The muscle isozyme is potently activated by either phosphorylation or AMP. In contrast, the brain isozyme is poorly activated by phosphorylation and its phosphorylated a form is more sensitive to AMP activation when enzyme activity is measured in substrate concentrations and temperatures encountered in the brain. The nonphosphorylated b form of the brain isozyme also differs from the muscle isozyme b form in its stronger affinity and lack of cooperativity for AMP. To identify the structural determinants involved, six enzyme forms, including four chimeric enzymes containing exchanges in amino acid residues 1-88, 89-499, and 500-842 (C terminus), were constructed from rabbit muscle and human brain phosphorylase cDNAs, expressed in Escherichia coli, and purified. Kinetic analysis of the b forms indicated that the brain isozyme amino acid 1-88 and 89-499 regions each contribute in an additive fashion to the formation of an AMP site with higher intrinsic affinity but weakened cooperativity, while the same regions of the muscle isozyme each contribute to greater allosteric coupling but weaker AMP affinity. Kinetic analysis of the a forms indicated that the amino acid 89-499 region correlated with the reduced response of the brain isozyme to activation by phosphorylation and the resultant increased sensitivity of the a form to activation by saturating levels of AMP. This isozyme-specific response also correlated with the glycogen affinity of the a forms. Enzymes containing the brain isozyme amino acid 89-499 region exhibited markedly reduced glycogen affinities in the absence of AMP compared to enzymes containing the corresponding muscle isozyme region. Additionally, AMP led to greater increases in glycogen affinity of the former set of enzymes. In contrast, phosphate affinities of all a forms were similar in the absence of AMP and increased approximately the same extent in AMP. The potential importance of a number of isozyme-specific substitutions in these sequence regions is discussed.


INTRODUCTION

For key enzymes of many metabolic pathways in eukaryotes, divergent evolution has spawned isozymic forms that tailor enzyme function to the specific environment and requirements of differentiated cells and tissues. In the case of glycogen phosphorylase, an enzyme documented by detailed structural analyses, isozyme forms with differential regulatory responses may provide a rich resource of insight into the details of the allosteric regulatory mechanisms.

Glycogen phosphorylase (EC 2.4.1.1) is a complex allosteric enzyme that catalyzes the regulated phosphorylysis of glycogen into glucose 1-phosphate (reviewed in Refs. 1-3). In mammals, phosphorylase exists as a family of three isozymes, muscle (M),() brain (B), and liver (L), named in association with the tissues where they predominate. All three isozymes exist as homodimers containing identical subunits of approximately 100 kDa and are encoded by distinct but structurally related genes situated on separate chromosomes in both the mouse (4) and human (1) . During development, the B isozyme predominates in fetal tissues, but is replaced partially or completely around birth by a distribution of the different isozymes (5, 6) . In the adult, each isozyme exhibits its own distinct pattern of widespread, but regulated expression across tissues (5, 7, 8, 9, 10) .

Detailed kinetic analysis, carried out primarily with the rabbit M isozyme, has shown that phosphorylase activity is intricately regulated (1-3). The enzyme is activated by intracellular ligands, such as AMP and glycogen, which upon binding promote active conformers. In contrast, glucose, glucose 6-phosphate, and purine nucleosides inhibit enzyme activity by stabilizing inactive conformers. X-ray crystallographic analysis of the rabbit M isozyme bound to a variety of ligands (11, 12, 13, 14, 15, 16, 17, 18) indicates that AMP, glycogen, glucose, and purine nucleosides bind to distinct sites on the enzyme, while the glucose 6-phosphate site overlaps that used for AMP binding (11, 12, 18) . Specific covalent phosphorylation at Ser-14 also activates the enzyme and is controlled by extracellular hormonal factors regulating phosphorylase kinase and phosphorylase phosphatase activity (1, 2, 3) .

Less is known about allosteric control of the B isozyme. However, interesting differences in the activation properties of the rabbit B and M isozymes have been observed. While the M isozyme responds strongly to activation by both phosphorylation and AMP, the B isozyme responds less well to activation by phosphorylation when assayed at physiological temperatures and substrate concentrations encountered in the brain (19) . AMP is required for full activation of the phosphorylated a form and increases activity to a level more comparable to that of the M isozyme a form. The AMP binding affinity of the nonphosphorylated b form of the B isozyme is stronger than that of the M isozyme (19, 20) and while the M isozyme binds AMP with high cooperativity, AMP binding in the B isozyme is noncooperative (20) . Finally, the glycogen affinity of the B isozyme is reduced compared to that of the M isozyme, but is markedly increased in the presence of AMP (19) .

The three-dimensional structure of the rabbit M isozyme reveals the AMP binding site and Ser-14-P in close proximity to each other. Both AMP and phosphorylation are thought to activate the enzyme by a similar overall mechanism (11, 12, 16, 17, 21) involving complex tertiary and quaternary changes within the ``activation subdomain'' comprising amino acid residues (aa) 1-120 (17, 22, 23) . It is therefore of interest to consider how differential responses to activation by phosphorylation and AMP arise in the B and M isozymes. In order to assess the role of structural differences between these isozymes, we have used rabbit M and human B phosphorylase cDNAs to construct a set of chimeric enzymes containing exchanges in the regions aa 1-88, 89-499, and 500-842 (C terminus). The enzymes encoded by these cDNAs were expressed in high amounts in Escherichia coli and purified for kinetic analysis.


EXPERIMENTAL PROCEDURES

Construction of Domain Exchanges between M and B Phosphorylase Isozymes

Rabbit M phosphorylase cDNA containing the entire protein coding region was inserted previously (24) into the pTACTAC expression vector (25) at the unique NdeI and HindIII sites downstream of the TACTAC promoter. cDNA containing the entire protein coding region of human B phosphorylase was obtained by ligating two overlapping partial cDNAs (7) at a common NcoI site at aa 792. To subclone the full-length B phosphorylase cDNA into the pTACTAC vector, NdeI and HindIII restriction sites were engineered into the 5`- and 3`-untranslated regions of the cDNA, respectively, by oligonucleotide-directed mutagenesis using standard protocols (26) .

M and B phosphorylase cDNAs containing exchanges in amino acid coding regions aa 1-88, 89-499, and 500-C terminus were constructed as shown in Fig. 1. To simplify nomenclature, enzymes are referred to by a three-letter designation corresponding to the M or B isozyme origin of the above three regions.


Figure 1: Construction of phosphorylase cDNAs for BMM, MBB, MBM, and BBM containing exchanges in amino acid regions aa 1-88, 89-499, and 500-842 (C terminus).



For construction purposes, an EcoRI site was introduced at aa 88 in MMM cDNA and an NheI site was introduced into both BBB and BMM cDNAs at aa 499 without changing the primary amino acid sequence. This was accomplished by site-directed mutagenesis (24, 27) using the following synthetic oligonucleotide primers, where the asterisk indicates a mismatch.Single-stranded cDNA templates were obtained using the Bluescript vector KSm (Stratagene), which had been modified to contain a NdeI site in the polylinker.

Chimeric phosphorylase cDNAs were generated by restriction endonuclease digestions, followed by ligation to exchange specific cDNA regions. Prior to transformation, unwanted ligation products were reduced by linearization with selective restriction endonuclease digestions (Fig. 1). All cDNA constructs were identified and verified following transformation by diagnostic restriction endonuclease digestions.

Expression of Phosphorylase cDNAs in E. coli

Phosphorylase cDNAs were expressed in E. coli as described previously (24) . E. coli 25A6 cells (W3110; tonA,lonD,galE,htpP) containing phosphorylase cDNAs in the pTACTAC vector were grown overnight in LB/amp (Luria broth plus 50 µg/ml ampicillin). Cells were then pelleted, resuspended in the same volume of fresh LB/amp and inoculated into a 40-fold greater volume of expression culture medium containing LB/amp, 250 µM isopropyl-1-thio--D-galactopyranoside (Sigma), 3 mM MnCl, and 0.5 mM pyridoxine (Sigma). The culture was grown for 48-50 h at 22 °C to allow high phosphorylase expression. Cells were then pelleted, quick frozen in liquid nitrogen, and stored at -70 °C.

Purification of Phosphorylase b Forms

Preparation of Cell Extracts

E. coli 25A6 cell pellets were resuspended in 1/12.5 of the original expression culture volume in homogenization buffer containing 25 mM -glycerophosphate, pH 7.0, 250 mM NaCl, 0.3 mM -mercaptoethanol, 0.3 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride (Sigma), 0.7 µg/ml pepstatin A (Boehringer Mannheim), 0.5 µg/ml leupeptin (Boehringer Mannheim), and 0.01% (w/v) benzamidine HCl (Sigma). Cell extracts were generated either by sonication or with a French press and clarified by centrifugation at 16,000 g for 45 min, followed by filtration through a 0.45-µm filter (Nalgene).

Immobilized Metal Affinity Column Chromatography

Cell extracts were fractionated by copper affinity column chromatography as described previously (28) . Briefly, cell extracts were loaded onto columns of chelating Sepharose (Pharmacia Biotech Inc.) equilibrated first with 50 mM CuCl and then with 1 mM imidazole (Sigma) in 25 mM -glycerophosphate, pH 7.0, and 250 mM NaCl. Phosphorylases were then eluted with a linear gradient of 1-100 mM imidazole and collected into tubes containing EDTA and -mercaptoethanol to make a final concentration of 0.5 mM each. Fractions containing phosphorylase activity were pooled, dialyzed against 25 mM -glycerophosphate, pH 7.0, 0.3 mM EDTA, 0.3 mM -mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 0.01% (w/v) benzamidine HCl, and 10% (v/v) glycerol.

DEAE-Sepharose Column Chromatography

For further purification, phosphorylases were dialyzed against 25 mM -glycerophosphate, pH 6.8, 1 mM EDTA, 1 mM -mercaptoethanol and loaded onto DEAE-Sepharose Fast Flow (Pharmacia) columns equilibrated with the same solution. Fractionation by DEAE-cellulose chromatography has been used previously to separate and purify rabbit M and B isozymes (20) . The phosphorylase b forms in this analysis were eluted from DEAE-Sepharose with a linear gradient of 0 M NaCl/25 mM -glycerophosphate, pH 6.8 to 250 mM NaCl/25 mM -glycerophosphate, pH 7.2. Fractions containing phosphorylase activity were pooled, concentrated with a Centriprep 10 concentrator (Amicon), and stored at -20 °C in 50% glycerol.

In Vitro Phosphorylation of Phosphorylases

Conversion of phosphorylases from the b to a forms was carried out enzymatically with rabbit muscle phosphorylase kinase, a gift from Dr. Donal Walsh, University of California, Davis (29) . Phosphorylation was carried out in 0.2 mM ATP, 5 mM MgCl, 1 mM CaCl, 100 mM Tris-HCl, pH 8.5, at 30 °C. Preliminary test conversions were performed with [-P]ATP (1100 cpm/pmol) to determine the optimal incubation time for phosphorylation of each phosphorylase construct. Incorporation of P into phosphorylase was determined by a filter paper assay (30).

DEAE-Sepharose Purification of Phosphorylase a Forms

Following conversion, a forms of phosphorylases were further purified by DEAE-Sepharose column chromatography. Prior to chromatography, phosphorylases were diluted 20-fold in DEAE-Sepharose loading buffer consisting of 25 mM -glycerophosphate, pH 6.8, 1 mM EDTA, and 1 mM -mercaptoethanol. Following loading in the above buffer, phosphorylases were eluted with a linear gradient of 50 mM NaCl/25 mM -glycerophosphate, pH 6.8, to 250 mM Nacl/25 mM -glycerophosphate, pH 7.2. Conditions for concentration and storage of the a forms were identical to those described for the b forms.

Slot-blot Assay for Phosphorylase Activity

Phosphorylase activity in column fractions was determined in the direction of glycogen formation by a rapid slot-blot assay. Approximately 100 µl of column fractions were slot-blotted onto a nitrocellulose filter (Bio-Rad) presoaked for 10 min in 50 mM -glycerophosphate, pH 7.0, and 0.1% glycogen (bovine liver type IX, Sigma). Following sample loading, the filter was incubated for 0.5-1 h at room temperature in 25 mM -glycerophosphate, pH 7.0, 1% (w/v) glycogen, 75 mM glucose 1-phosphate, and 1 mM AMP. The extent of glycogen formation on the filter was determined with iodine stain (0.4% KI plus 0.2% I).

Kinetic Analysis

In order to determine kinetic parameters of AMP activation of the phosphorylase b forms, a modification of the procedures of Carney et al.(31) was used to assay phosphorylase in the direction of glycogen synthesis. Assays at each condition were done in triplicate. The enzymes were preincubated for 5 min at 30 °C in 10 mM BES, pH 6.8, 1 mM EDTA, 1 mM dithiothreitol in the presence of 1% glycogen (equivalent to 62 mM glucose residue units) and varying concentrations of AMP. The reaction was initiated by addition of glucose 1-phosphate to a concentration of 30 mM. At the end of the reaction, 50% trichloroacetic acid was added to a final concentration of 5% and the reaction mixtures were placed on ice. The colorimetric detection of phosphate was used to measure the amount of phosphate generated by the conversion of glucose 1-phosphate into glycogen (31) . Reaction aliquots were added to the color reagent and incubated exactly 5 min at room temperature before measurement of 316 nm absorbance.

For kinetic analysis of phosphorylase a forms, activity was assessed in the direction of glycogenolysis by a P assay originally developed by Gevers and Stalmans (32) and modified for small volumes as described previously (33) . Phosphorylase was incubated for 10 min at 37 °C in a 125-µl final volume of reaction mixture containing varying concentrations of potassium phosphate, pH 7.4, 1 µCi of [P]dipotassium monohydrogen phosphate (1.0 Ci/mmol, DuPont NEN) and varying concentrations of bovine liver glycogen (type IX, Sigma) and AMP. Prior to assay, the [P]dipotassium monohydrogen phosphate was incubated for 2 h at 100 °C with 4.5 volumes of 1 N HCl to hydrolyze polyphosphates and then neutralized with NaOH. The phosphorylase reaction was terminated by adding 100 µl of the reaction mixture to 25 µl of 4.0 M perchloric acid plus 2 µl of 1.0 M nonradioactive potassium phosphate, pH 7.4, at 4 °C. A 475-µl solution of 31.7 mM ammonium molybdate and 52.6 mM triethylamine, pH 5.0, was then added, and the mixture was incubated at 4 °C for a minimum of 30 min to precipitate unincorporated phosphate. Following centrifugation at 12,000 g for 30 min, 400 µl of the supernatant was transferred to 6 ml of Biofluor (DuPont) for liquid scintillation analysis. With this assay, one unit of phosphorylase activity is defined as the amount of enzyme required to convert 1 µmol of inorganic phosphate into ammonium molybdate-triethylamine-soluble material per min. For specific activity determinations, protein concentration was measured by the Bradford procedure (34) . Values obtained were used in nonlinear least squares fitting (JMP version 2 software, SAS Institute) to determine refined estimates of kinetic parameters.


RESULTS

Phosphorylase Chimeras

Four chimeric phosphorylases, BMM, MBB, MBM, and BBM, were constructed by exchanging rabbit M (MMM) and human B (BBB) phosphorylase cDNA regions encoding aa 1-88, 89-499, and 500-C terminus. The three-letter designation of these enzymes corresponds to the M or B isozyme origin of these regions in the N- to C-terminal direction. It should be noted that DNA constructs for chimeras BMB and MMB were also prepared, but we were unable to express enzymes in sufficient quantity for analysis.

The location of these regions in the three-dimensional structure of MMM is presented in Fig. 2(A and B). The region aa 1-88 (see Fig. 2A) contains the majority of residues in the activation subdomain (aa 1-120), including the N-terminal tail (aa 1-23), which rotates to create a number of intersubunit contacts following phosphorylation (12, 16, 17) , residues Arg 43` and Arg 69 which are involved in Ser-14-P binding (12) , and residues in the CAP loop (aa 41`-47`) and helix-2 (aa 48-78), which comprise part of the AMP binding site (11, 12, 16) . (The prime symbol denotes residues in the diad-related subunit.) The second exchanged region, aa 89-499, makes up the rest of the N-terminal domain and contains residues in helix-8 (aa 290-312) and the aa 313-325 loop that comprise the remainder of the AMP binding site (11, 12, 16, 17) , as well as residues of the tower helix aa 262-277 that form intersubunit contacts at the catalytic face of the dimer (see Fig. 2B). This region also contains residues within regions aa 205-215 and 354-362 and residues within aa 387-438 which comprise the minor and major glycogen storage sites, respectively (13, 14) . The third exchanged region, aa 500-C terminus, comprises the C-terminal domain (1, 2) which contains the majority of residues which make up the active site and pyridoxal phosphate cofactor binding site. The active site itself is situated in a cleft between the N- and C-terminal domains.


Figure 2: The location of exchanged regions between the M and B isozymes in relation to the three-dimensional structure of the rabbit M isozyme a form dimer complexed with glucose (Ref. 12; S. R. Sprang, E. J. Goldsmith and R. J. Fletterick, unpublished results). Exchanged regions are colored as mediumgray for aa 1-88, lightgray for aa 89-499, and darkgray for aa 500-C terminus. A, side view of the enzyme highlighting the aa 1-88 exchanged region within the activation subdomain. The locations of the phosphorylation sites at Ser-14 and the AMP binding sites in both subunits and the glycogen storage site with maltoheptaose bound in the lower subunit are shown. B, front view of the catalytic face of the dimer showing the two active site clefts (containing glucose, phosphate, and the cofactor pyridoxal phosphate in side view) located between the exchanged regions, aa 89-499 and 500-C terminus. The glycogen storage sites with malto- heptaose bound are shown in the aa 89-499 exchanged region in both subunits. In this orientation, the activation subdomain is in the back of the molecule.



Phosphorylase Purification

The following general scheme was developed for the expression and purification of all phosphorylases. Phosphorylase b forms were expressed in E. coli from cDNAs inserted in the pTACTAC vector under the control of the double trp-lac promoter. Following sonication of E. coli cells, phosphorylases were purified by chromatography on immobilized metal affinity and DEAE-Sepharose columns. An additional DEAE-Sepharose column fractionation was used to further purify in vitro phosphorylated a forms.

The initial immobilized metal affinity column fractionation led to higher purification of MMM and the chimeric phosphorylase, BMM, compared to BBB and the other chimeric enzymes. This was due, in part, to differences in elution behavior of these enzymes. As described previously (28) , MMM eluted at a higher concentration of imidazole (39 mM) than that required for elution of BBB (34 mM). BMM eluted similarly to MMM at 43 mM imidazole, and MBB eluted similarly to BBB at 35 mM imidazole, while MBM and BBM eluted at lower imidazole concentrations of 24 mM and 20 mM, respectively. Elution at higher imidazole concentrations led to greater removal of impurities in the E. coli cell sonicates and, hence, resulted in greater purification of MMM and BMM. Another factor contributing to their higher purity was that both MMM and BMM were present in higher amounts (3-10-fold) in E. coli sonicates compared to BBB and the other chimeric enzymes. The recovered amount of all phosphorylases, however, could be improved (approximately 3-fold) by disrupting E. coli cells with a French press.

On DEAE-Sepharose the b form enzymes eluted at different NaCl concentrations (45-110 mM) that correlated with estimated isoelectric points derived from their primary sequences. MMM (pI = 6.9) and BMM (pI = 6.6) eluted at the lowest NaCl concentrations, and this resulted in better removal of impurities than in the case of BBB (pI = 6.3) and the other chimeric enzymes (pI = 6.4, pI = 6.3, and pI = 6.2). In most cases, the a forms eluted at higher NaCl concentrations (100-140 mM) than the b forms. Hence, passage of the phosphorylase a forms over DEAE-Sepharose resulted in further purification. Lack of detectable phosphorylase b forms in the elution profiles confirmed the completeness of in vitro phosphorylation. All enzymes were greater than 90% pure based on SDS-polyacrylamide gel electrophoresis (35) , and maximum yields of all phosphorylases ranged from 10 to 40 mg/liter of starting E. coli culture.

M and B isozymes can be distinguished by their mobilities on native polyacrylamide gels containing 100 µM glycogen (5, 9). On native gels prepared as described previously (36) , the b form of BBB migrated faster than that of MMM, while all of the chimeric enzymes exhibited intermediate mobilities (data not shown). The mobility patterns of the chimeric enzymes correlated roughly with their estimated isoelectric points. Interestingly, all chimeric enzymes containing the aa 89-499 region from the B isozyme exhibited migration rates very similar to that of BBB, while BMM containing the corresponding region from the M isozyme exhibited a mobility similar to that of MMM. As the aa 89-499 region contains the glycogen storage site, the mobility patterns of these enzymes may be determined in part by differential enzyme affinity for glycogen in the gel.

AMP Affinity and Cooperativity of Phosphorylase b Forms

Enzymatic activities of the phosphorylase b forms were measured at varying concentrations of AMP in order to compare their activation responses to AMP binding. Refined estimates of kinetic parameters of AMP activation are summarized in . V, S(AMP), and Hill coefficient (n) were derived for each enzyme by nonlinear least squares fitting to the Hill equation (37) .

Based on the S value, BBB was found to bind AMP with about 2-fold stronger apparent affinity than MMM. BBB also exhibited noncooperative AMP activation (n = 1.14), while MMM exhibited high cooperativity (n = 1.75). The S values of the other enzymes fell within the range of the MMM and BBB values, indicating that the general activating response to AMP was retained in all enzymes and not compromised by the replacement of sequence regions.

Chimeric phosphorylases revealed interesting correlations between sequence regions and the cooperativity of AMP responses. When the C-terminal domain (aa 500-842) of the B isozyme was replaced by that of the M isozyme, the resulting enzyme, BBM, displayed the same noncooperative AMP response as BBB. Converting MBB into MBM would involve a similar exchange of C domains, and these enzymes, within experimental error, also did not differ significantly in AMP cooperativity. This suggests that the property of AMP cooperativity is invariant with respect to the origin of the C domain.

Comparing enzymes that differ only in the origin of the aa 89-499 region indicated that replacing the muscle isozyme aa 89-499 with the brain isozyme aa 89-499 region (MMM MBM, BMM BBM) leads to lower cooperativity, suggesting that sequence substitutions in the aa 89-499 region play a role in determining AMP cooperativity. This is further supported by the finding that BMM was as highly cooperative as MMM and even exhibited stronger apparent AMP affinity. In fact, the BMM data plus the observation that AMP response is not significantly affected by swapping C domain regions (aa 500-842) would sufficiently indicate that the aa 89-499 region strongly governs differential AMP cooperativity in the M and B isozymes.

While exchanging the aa 1-88 region in MMM BMM conversion had no effect on AMP cooperativity, the same exchange in the cases of BBB MBB and BBM MBM did produce a change in cooperativity. When the M aa 1-88 region replaced the B aa 1-88 region in either of the noncooperative enzymes BBB or BBM, the resulting enzyme exhibited increased cooperativity. Thus, it would appear that the M isozyme aa 1-88 region also confers properties of AMP cooperativity, although it is not sufficient to effect the full cooperativity of the native muscle isozyme.

Comparison of Intrinsic AMP Affinity in the Phosphorylase b Forms

While differences in S provide a general comparison of AMP affinity, S values are an unreliable indicator of the nature of the AMP binding site in the unbound phosphorylase dimer, particularly when strong homotropic interactions exist between the two sites. Therefore, the activity data was also analyzed to determine the apparent intrinsic dissociation constant for AMP, K`, as this provides a better structural measure of how the sequence exchanges affect the formation of a native unbound AMP site. presents estimates of the apparent intrinsic AMP affinity and interaction coefficient, , obtained by fitting a velocity equation derived previously (38) .

The apparent intrinsic AMP affinity of BBB was about 10-fold stronger than that of MMM in this analysis. Values of K` for the muscle isozyme from previous studies would suggest an even larger difference between MMM and BBB (19, 20). Comparisons between BBB and BBM or between MBB and MBM indicate that exchanging the C-terminal aa 500-842 region has no effect upon K`. On the other hand, replacing an M aa 1-88 region with a B aa 1-88 region leads to a significant change of K`. In all three cases represented in our test set (MBB BBB, MBM BBM, MMM BMM), K` decreased, indicating that resulting enzymes formed an AMP site with stronger binding affinity for the first molecule of the activating ligand. Thus, substitutions in the aa 1-88 region between brain and muscle phosphorylases play a determining role in the affinity of the site. From similar comparisons for the effect of exchanging the aa 89-499 regions, the data suggest that substitutions in the aa 89-499 region also affect the affinity of the AMP site. Again, the effect observed is that changing a muscle aa 89-499 region into a brain aa 89-499 results in an AMP site with improved affinity.

The difference in the apparent free energy of binding between the brain and muscle AMP binding sites is about 1.4 kcal/mol, as determined from the expression shown in Equation 1.

On-line formulae not verified for accuracy

Similar determinations using the apparent intrinsic dissociation constants for the other enzymes indicate that the brain isozyme aa 1-88 and 89-499 regions contribute equally (0.6-0.8 kcal/mol) and in an additive manner to the overall free energy change.

The Effect of Phosphorylation on Enzyme Activity

The effect of phosphorylation in the absence or presence of saturating levels (100 µM) of AMP on the activities of these enzymes was determined at physiological temperature (37 °C) and substrate concentrations (2 and 5 mM glycogen, 10 mM phosphate) that approximate conditions in the brain (19) . Enzyme activity was also assessed at high glycogen concentration (50 mM). Specific activity values are presented in . In agreement with previous analysis of the rabbit B isozyme (19) , the specific activity of the a form of BBB was low when determined in the absence of AMP and in 2 and 5 mM glycogen. Activity, however, increased dramatically (7-9-fold) in 100 µM AMP. In contrast, the specific activity of the a form of MMM minus AMP was much higher (6-8-fold) than that of BBB minus AMP and the degree of enzyme activation by AMP was lower (3-4-fold). This led to specific activity values for MMM that were 2-3-fold higher in AMP than those for BBB. Replacing aa 1-88 in MMM with the corresponding B isozyme region (i.e. BMM) did not appreciably change the specific activity of the enzyme minus AMP or the extent of enzyme activation by AMP. However, all chimeric enzymes containing the aa 89-499 region of BBB (MBB, MBM, and BBM) behaved more like BBB. These enzymes exhibited the same low levels of activity as BBB in the absence of AMP, and the degree of enzyme activation by AMP was intermediate (4-7-fold). In AMP, the activity of these ``xBx'' chimeras was similar to or slightly lower than that of BBB in AMP. Hence, isozymic differences in activation by phosphorylation and in the degree of enzyme activation of the a forms by saturating levels of AMP correlate with the isozymic origin of the aa 89-499 region. Exchanges in the C-terminal domain (aa 500-C terminus) do not correlate with these isozymic differences since BBB/BBM and MBB/MBM pairs all exhibited similar responses to activation by phosphorylation and AMP. Isozymic differences also do not correlate with the aa 1-88 region since the response of BMM was similar to that of MMM, while MBB responded like BBB and MBM like BBM.

Glycogen is both a substrate and an activator of phosphorylase (1) . Increasing the glycogen concentration to 50 mM in the assay led to a much reduced AMP effect (approximately 2-fold) on all enzyme activities. For enzymes containing the B isozyme aa 89-499 region, this reduced activation by AMP was due primarily to an increase in their specific activities in the absence of AMP. In contrast to enzyme activation by AMP, changes in glycogen concentration in the physiological range (2 and 5 mM) did not lead to dramatic changes in enzyme activity either in the absence or presence of AMP.

In order to distinguish between the separate effects of phosphorylation and AMP on enzyme activation, nonphosphorylated b forms were assayed under similar conditions (data not shown). In 5 mM glycogen, all phosphorylase b forms exhibited specific activities in the range of 0.5-1.5 units/mg in the presence of 100 µM AMP and 200-fold less activity in the absence of AMP. Phosphorylated MMM and BMM exhibited 5-fold higher specific activities than their corresponding AMP-activated b forms, while BBB and chimeric enzymes with the B isozyme aa 89-499 region were activated to similar extents whether by phosphorylation or by 100 µM AMP. These findings again demonstrate the relative insensitivity of BBB to activation by phosphorylation and indicate its correlation with the aa 89-499 region.

Glycogen Affinity of a Forms in the Absence and Presence of AMP

Since the differential effect of phosphorylation and 100 µM AMP on the activation of the b and a forms, respectively, of the M and B isozymes correlated with the aa 89-499 region and occurred only in moderate, physiological glycogen concentrations, we considered whether isozyme-specific differences in glycogen affinity determine the responsiveness of these enzymes to phosphorylation and AMP. K values for glycogen were therefore assessed in all phosphorylase a forms in the absence and presence of AMP. It should be noted that glycogen binds to three distinct sites on a phosphorylase monomer. Two of these together form the so-called glycogen storage site (13, 14) , while the third locus is the active site itself. It has been shown previously that the affinity of MMM for glycogen is at least 20-fold higher at the glycogen storage site than at the active site (39) . Hence, the K values determined here probably reflect affinity for glycogen at the glycogen storage site. The function of the glycogen storage site is presumed to be 2-fold, to target the enzyme to its substrate for localization and to promote substrate binding at the active site (1, 13, 14) .

Michaelis-Menten constants for glycogen, K (glyc), determined for the a forms in the absence of AMP and at different, fixed phosphate concentrations are presented in I. All K (glyc) values decreased with increasing phosphate concentration. Interestingly, K (glyc) values for BBB were much higher (5-6-fold) than those for MMM when determined in concentrations of phosphate, 5 and 10 mM, that are in the physiological range (1-10 mM). BMM exhibited K (glyc) values similar to those of MMM, while the enzymes, MBB, MBM, and BBM, containing the B isozyme aa 89-499 region, exhibited K (glyc) values intermediate to those of MMM and BBB.

K (glyc) values for the a forms were also determined in the presence of 100 µM AMP and are shown in . In physiological (2-10 mM) phosphate concentrations, the K (glyc) values for BBB decreased dramatically (12-20-fold) in AMP and were now more similar to MMM, being only 2-3-fold higher. K (glyc) values for MMM were less responsive to AMP, decreasing 3-7-fold in 5-10 mM phosphate. BMM exhibited K (glyc) values similar to those of MMM, while all enzymes containing the B isozyme aa 89-499 region, MBB, MBM, and BBM, exhibited K (glyc) values similar to those of BBB. Hence, isozyme-specific differences in the glycogen affinities of the a forms of MMM and BBB determined in the absence and presence of 100 µM AMP show significant correlation with the aa 89-499 region of the protein, but not with the regions containing aa 1-88 or 500-C terminus.

Phosphate Affinity of a Forms in the Absence and Presence of AMP

The affinities of the a forms for the second substrate, phosphate, K (P), were also determined at 37 °C in the absence and presence of 100 µM AMP. Values are presented in Tables V and VI, respectively. In the absence of AMP and in 10 mM glycogen, K (P) values for all phosphorylases were approximately the same although enzymes with the B isozyme aa 89-499 region exhibited a significant degree of homotropic cooperativity making exact comparisons difficult. For the latter enzymes, data points were fit to the Hill equation (37) to obtain estimates of the degree of cooperativity and K (P) values. Homotropic cooperativity of MMM and BMM for phosphate was observed at lower concentrations of glycogen (2-5 mM). In higher glycogen concentrations (25-100 mM), K (P) values for enzymes with the B isozyme aa 89-499 region decreased approximately 2-fold and cooperativity was reduced (data not shown).

Homotropic cooperativity was not observed in the M, B, and chimeric enzymes when K (P) values were determined in AMP. In 100 µM AMP and 10 mM glycogen, K (P) values decreased 5-10-fold compared to the values determined minus AMP. In these conditions, K (P) values were approximately 2-fold higher for BBB and enzymes containing the B isozyme aa 89-499 region compared to MMM and BMM, which exhibited similar K (P) values. In lower glycogen concentrations (0.5-5 mM), K (P) values for all enzymes increased slightly, up to 2-3-fold.


DISCUSSION

In the absence of inhibitors, the activity reached by phosphorylases in vivo depends on the AMP level, the degree of phosphorylation and the concentration of substrates. In this report, we have used chimeric enzymes to identify regions in the protein that govern the differential responses of the M and B isozymes to activation by phosphorylation and AMP.

These studies indicate that differential cooperativity of AMP activation and apparent intrinsic AMP affinity of the b forms are governed in an additive fashion by both the aa 1-88 and 89-499 regions of the protein. The brain aa 1-88 and 89-499 regions each contribute to forming an improved (i.e. high affinity) AMP site and weakened cooperativity, while the same regions of the M isozyme each contribute to greater allosteric coupling but a weaker AMP site.

In contrast, the differential response of the M and B isozymes to activation by phosphorylation and the resultant differential sensitivities of the a forms to saturating levels of AMP correlate solely with the aa 89-499 region of the protein. Based on analysis of substrate affinities, this is probably due to significant differences in the glycogen affinities of the a forms which also correlate with the aa 89-499 region. Verifying a previous report (19), this differential response is most pronounced when activities are determined in physiological temperatures and substrate concentrations encountered in the brain. Under these conditions in the absence of AMP, the glycogen affinity of the B isozyme was markedly lower than that of the M isozyme and K (glyc) values were approximately 5-10-fold higher than physiological glycogen concentrations (2-5 mM). Saturating levels of AMP, however cause glycogen affinity of the B isozyme to increase by an order of magnitude, bringing K (glyc) into the physiological range and closer to the K (glyc) of the M isozyme in AMP. Chimeras containing the B-isozyme aa 89-499 region exhibited glycogen affinities more similar to those of the B isozyme, while BMM, containing the M isozyme aa 89-499 region, exhibited glycogen affinities similar to those of the M isozyme. In contrast to their marked differences in glycogen affinity, M, B, and chimeric enzymes exhibited similar affinities to phosphate in the absence of AMP. AMP increased phosphate affinities 5-10-fold. For all enzymes, K values were slightly higher or within the physiological range of phosphate concentration (19) .

As a basis for identifying structural elements that govern differential allosteric responses of phosphorylase isozymes to activation, primary sequence comparisons of M, B, and L isozymes have been carried out in relation to the three-dimensional structure and function of the rabbit M isozyme (40, 41) . These studies indicate that there is very high conservation of residues in the active site and pyridoxal phosphate cofactor binding site, as well as high conservation of the secondary structural elements containing these residues. High conservation of these regions has also been noted for seven nonmammalian phosphorylases (42). Hence, differences between the M and B isozymes would not be expected to be related directly to differences in active site structure or affinity for substrates at the active site.

In the aa 1-88 region, 13 amino acid substitutions occur between the rabbit M and human B isozymes. Eleven of these are clustered in the first 30 residues, and eight, located throughout the region, have been designated as B isozyme-specific as they occur in B, but not M and L isozymes regardless of species (41) . While all of the residues implicated in intersubunit contacts in this region are exactly or highly conserved, it is reasonable to expect conformational rearrangement in the N-terminal tail as a summative consequence of many substitutions. The N-terminal tail upon activation may rotate and fold into a crevice between the two subunits, creating new intersubunit contacts that transmit conformational changes to residues of the other subunit (12, 16, 17) . Three consecutive, B isozyme-specific substitutions, V(M isozyme)21L(B isozyme), E22G, and N23D, clustered at the hinge of the N-tail rotation, could alter the tail movement, thereby affecting intersubunit interactions and the coupling of AMP sites. Other candidates for affecting AMP affinity and cooperativity are B isozyme-specific Y52F and K77R substitutions in helix-2 (aa 48-78), which form part of the dimer interface at the AMP locus.

In the aa 89-499 region, there are 54 amino acid differences between the rabbit M and human B isozymes, 29 of which are B isozyme-specific (41). The major structural components in this region that participate in AMP binding and allosteric activation are the tower helix (aa 262-277) and adjacent loops, both involved in intersubunit interactions, and a region at the end of helix-8 (aa 309-325) involved in AMP binding. Three B isozyme-specific substitutions, F196Y, N253K, and Q264E, may alter AMP cooperativity since they occur at or next to residues that are involved in dimer contacts. An additional substitution, G261D, may also be important since Asp-261 is present in both the B and L isozymes regardless of species and the L isozyme is known to exhibit noncooperative AMP activation (1) . In addition, G261D and Q264E may alter helix-helix stability and interactions of the tower helices. M isozyme mutations that alter tower helix interactions have been shown to significantly alter AMP cooperativity and binding (43) . Two other B isozyme substitutions, C171V and G172D, may also be important since they occur in a structural element, the Dali loop, that is thought to be involved in allosteric activation of the rabbit M isozyme (2) . The aa 265-324 stretch of residues is exactly conserved between B and M isozymes and includes the aa 313-325 loop, which is an important AMP binding determinant governing AMP/IMP specificity (17) . The nearest B isozyme-specific substitutions are an adjacent clustered set, N325C, D327E, and A328T, which may produce a slight conformational rearrangement of this loop.

With regard to the reduced affinity of the B isozyme a form for glycogen, 12 B isozyme-specific substitutions in the aa 89-499 region lie within the minor and major glycogen storage sites (41) . The majority of these do not occur at sugar binding residues, and of those that do, the amino acid change is generally conservative. However, one substitution at aa 405 has been noted as being potentially significant (41) since it is highly nonconservative (Glu to Ala) and occurs at an amino acid residue in the rabbit M isozyme, Glu-405, that interacts with multiple sugar residues (S6-S9) in the major glycogen storage site (13, 14) . A second nonconservative, B isozyme-specific substitution (Ala to Asp) also occurs in the major glycogen storage site at aa 435 (41) . Although Ala-435 in the rabbit M isozyme does not interact directly with sugar residues, the B isozyme substitution may be significant since it occurs in a flexible loop between two sugar binding residues, Glu-433 and Lys-437, which moves upon oligosaccharide binding to maximize sugar contacts (13, 14) .

In contrast to substitutions in the glycogen storage site that might directly affect glycogen affinity, differences in glycogen affinity might also arise from isozyme differences in the ability of the enzyme to respond properly to activation by phosphorylation. In this case, weaker glycogen affinity obtained from phosphorylation at Ser-14 might have been expected to be due to changes in the N-terminal, aa 1-88 region since the phosphoserine and its binding site are located in this sequence (1) . However, the transmission of energy from binding of the phosphopeptide N-terminal tail also involves the N-terminal domain up to aa 482 (12) . As mentioned above, substitutions at or near dimer contact residues or within the Dali loop may play a role in this regard.

Interestingly, the differential response of the M and B isozymes to activation by phosphorylation does not correlate with the N-terminal (aa 1-88) and C-terminal (aa 500-C terminus) regions of the protein. The aa 500-C-terminal domain of the protein, containing the majority of active site and pyridoxal phosphate binding site residues, is mobile during activation (23) . Hence, residues in this region could be involved in the enzyme's response to allosteric activation. The studies presented here, however, demonstrate that the aa 500-C-terminal region is not used to tailor the differential response. The N-terminal, aa 1-88 region forms about 40% of the intersubunit contacts and although substitutions in this region affect AMP coupling and affinity, they are not a determining factor for the differential response of these enzymes to activation by phosphorylation.

The difference in AMP and glycogen affinities of the phosphorylated M and B isozymes is relevant to their physiological roles. The B isozyme is prevalent in brain and fetal tissues (5, 6, 7, 8, 9, 10) and is thought to be responsible for providing an emergency energy source during brief periods of anoxia or hypoglycemia (1) . Hence, this isozyme would be expected to be more sensitive to intracellular control through changes in AMP concentration than extracellular control via phosphorylation. The low glycogen affinity of the B isozyme a form in the absence of AMP implies poor enzyme activity in vivo when AMP concentration is low. This would reduce glycogenolysis and therefore increase glycogen retention under levels of stress when the extracellular phosphorylation cascade system is active but internal energy charge is high. This would be especially important for fetal tissues and adult tissues such as the brain, which must maintain optimal glycogen levels for internal use. Poor glycogen affinity in the absence of AMP would also allow the a form to be more sensitive to AMP. The higher AMP affinity of the a and b forms of the B isozyme (19, 20) would also contribute to increased sensitivity to lower than saturating AMP concentrations. By comparison, in the 10-fold higher glycogen concentrations that occur in muscle, the a form of the M isozyme would be expected to be highly active and relatively insensitive to AMP. This would be in accord with the enzyme's physiological role of providing an internal energy source for muscle contraction (1) , since the enzyme must respond to extracellular neural and hormonal control via phosphorylation regardless of the internal energy state of the cell.

Our analysis of differential responses of the phosphorylated M and B forms underscores the importance of glycogen in the activation process of phosphorylase. In the B isozyme, the reduced glycogen affinity of the a form has tuned the enzyme so that the phosphorylated form in the absence of AMP is only weakly active at physiological glycogen concentrations, which are significantly below the K value. The greater responsiveness of the B isozyme a form to AMP arises because AMP improves glycogen affinity to the point where K and physiological glycogen concentration are similar. AMP also increases glycogen affinity of the phosphorylated M isozyme, but the effect has less physiological consequence because of the higher glycogen concentrations in muscle and the enzyme's higher affinity for glycogen in the absence of AMP.

  
Table: Comparison of phosphorylase b AMP cooperativity and affinity

n, Hill coefficient; S, concentration of AMP required for half-maximal activation; V, maximal enzyme velocity; , interaction coefficient; K`, apparent intrinsic AMP affinity (dissociation constant for first AMP ligand bound to enzyme dimer).


  
Table: Comparison of phosphorylase a specific activities in 10 mM phosphate and varying concentrations of glycogen, plus and minus AMP


  
Table: 1952671790p4in K (mM) ± standard error.(119)

  
Table: Estimates of K (glycogen) of phosphorylase a forms assayed in 100 µM AMP and varying phosphate concentrations


  
Table: 1953439843p4in n, Hill coefficient.(119)

  
Table: Estimates of K (P ) for phosphorylase a forms assayed in 100 µM AMP and varying glycogen concentrations



FOOTNOTES

*
This work was supported by Natural Sciences and Engineering Research Council of Canada Grant A8383 (to M. M. C.) and Grant DK32822 from the National Institutes of Health (to R. J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Biology, York University, 4700 Keele St., North York, Ontario M3J 1P3, Canada.

To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0448.

The abbreviations used are: M, muscle; B, brain; L, liver; aa, amino acid(s); BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid.


ACKNOWLEDGEMENTS

We are grateful to Dr. John Hudson for assistance in the construction of plasmids and to Arnold Chin for technical assistance in the purification of enzymes.


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