TIBS 17 - FEBRUARY 1992
Council and The G6ran Gustafsson Foundation for financial support. We wish to thank our many colleagues who have worked with us o n various properties of photosystem II and its photoinactivation and in particular, Steven
Hall and Hugh Salter for helping us prepare the figures used in this article.
References 1 Kyle, D. J., Osmond, C. B. and Arntzen, C.J. (1987) Photoinhibition, Topics in Photosynthesis (Barber, J., ed.) (Vol. 9), Elsevier 20smond, C. B. and Chow, W. S. (1988) Aust. J. Plant PhysioL 15, 1-9 3 Ewart, A. J. (1896) J. Linn. Soc. 31, 364-461 4 Andersson, B. and Styring, S. (1991) in Current Topics in Bioenergetics (Lee, C. P., ed.) (Vol. 16), pp. 1-81, Academic Press 5 Anderson, J. M. and Andersson, B. (1988) Trends Biochem. Sci. 13, 351-355 6 Kyle, D. J., Ohad, I. and Amtzen, C. J. (1984) Proc. Natl Acad. Sci. USA 81, 4070-4071 7 Mattoo, A. K., Marder, J. B. and Edelman, M. (1989) Cell 56, 241-246 8 Michel, H. and Deisenhofer, J. (1988) Biochemistry 27, 1-7 9 Trebst, A. (1986) Z. Naturforsch. 41c, 240-245 10 Barber, J. (1987) Trends Biochem. Sci. 12, 321-326 11 Rutherford, A. W. (1989) Trends Biochem. Sci.
GLYCOGEN PHOSPHORYLASE is an essential enzyme which, in mammals, provides the fuel needed to sustain life between feeding. Phosphorylase senses the metabolic state of the cell or organism, and responds by liberating glucose from stored glycogen as needed. Phosphorylase is studied both for its central role in an important biological process and also as a model for allosteric behavior in proteins, The activation and inhibition of phosphorylases are ceil, tissue and organism specific. In mammals, phosphorylase must be switched from its dormant state to the active form in order to liberate glucose 1-phosphate (Glc-I-P) from glycogen, with the help of inorganic phosphate 1. Inside muscle cells, an increase in the products of ATP hydrolysis, principally AMP, signals the enzyme to commence digesting glycogen. Activation of phosphorylase by AMP can be competitively inhibited by metabolic indicators of ample energy, such as glucose 6-phosphate.(Glc-6-P) and ATP. M. F. Browner and R. J. Fletterick are at the Department of Biochemistry and Biophysics, Box 0448, Universityof California, San Francisco, CA 94143-0448, USA.
66
14,227-232
Acta1060,1-12
12 Prasil, 0., Adir, N. and Ohad, I. (1992) in The Photosystems, Topics in Photosynthesis
(Barber, J., ed.) (Vol. 11), pp. 220-250, Elsevier 13 Eaglesham, A. R. J. and Ellis, R. J. (1974) Biochim.Biophys. Acta 335, 396-407 14 Edelman, M. and Reisfeld, A. (1978) in Chloroplast Development (Akoyunoglou,G. and
Argyroudi-Akoyunoglou,J. H., eds), pp. 641-852,
Elsevier
27 Schuster, G., Timberg, R. and Ohad, I. (1988) Eur. J. Biochem. 177, 403-410 28 Trebst, A., Depka, B. and Kipper, M. (1990) in Current Research in Photosynthesis
(Baltscheffsky, M., ed.) (Vol. 1), pp. 217-222, Kluwer Academic 29 Aro, E-M., Hundal, T., Carlberg, I. and Andersson, B. (1990) Biochim. Biophys. Acta
1019, 269-275
15 Greenberg, B. M. et al. (1989) Proc. Natl Acad. Sci. USA 86, 6617-8620 16 Strid, A., Chow, W. S. and Anderson, J.M. (1990) Biochim. Biophys. Acta 1020,
260-268
30 Virgin, I., Ghanotakis, D. F. and Andersson, B. (1990) FEBSLett. 269, 45-48 31 Virgin, I., Salter, H., Ghanotakis, D. F. and Andersson, B. (1991) FEBSLett. 287,
125-128
17 Setlik, I. et al. (1990) Photosynth. Res. 23,
39-48 18 Vass, I. et al. Proc. NatlAcad. Sci. USA (in
press) 19 Telfer, A., He, W-Z. and Barber, J. (1990) Biochim. Biophys. Acta 1017, 143-151 20 Shipton, C. A. and Barber, J. (1991) Proc. Natl Acad. Sci. USA 88, 6691-6695 21 Hundal, T., Aro, E. M., Carlberg, I. and Andersson, B. (1990) FEBS Lett. 267, 203-206 22 Kirilovsky, D. and Etienne, A-L. (1991) FEBS Lett. 279, 201-204 23 Telfer, A., De Las Rivas, J. and Barber, J. (1991) Biochim. Biophys. Acta 1060, 106-114 24 Callahan, F. E., Becker, D. W. and Cheniae, G.M. (1986) Plant Physiol. 82, 261-269 25 Jegerschold, C. and Styring, S. (1991) F E B S Lett. 280, 87-90 26 Mayes, S. R. et al. (1991) Biochim. Biophys.
32 Shipton, C. A. and Barber, J. Biochim. Biophys. Acta (in press) 33 Greenberg, B. M., Gaba, V., Mattoo, A. K. and Edelman, M. (1987) EMBOJ. 6, 2865-2869 34 Trebst, A. and Depka, B. (1990) Z. Naturforsch.
45c, 765-771 35 Barbato, R., Shipton, C. A., Giacometti, G. M. and Barber, J. (1991) FEBSLett. 290, 162-166 36 Krause, H. G. and Behrend, U. (1986) FEBS Lett. 200, 298-302 37 Demmig-Adam,B. (1990) Biochim. Biophys. Acta 1020, 1-24 38 Takahashi, Y., Hansson, 6 , Mathis, P. and Satoh, K. (1987) Biochim. Biophys. Acta 893,
49-59 39 Thompson, L. K. and Brudvig, G. W. (1988) Biochemistry 27, 6653-6658 40 Somersalo, S. and Krause, G. H. (1989) Planta
177, 409-416
Phosphorylase: a biological transducer
A transducer is a device that receives energy from one system and transmits it, often in a different form, to another. Glycogen phosphorylase receives information from the cell or organism in the form of metabolic signals. The energy associated with the binding of these ligand signals is integrated and transmitted at an atomic level, allowing precise adjustment of the enzymatic activity. Understanding this elegant allosteric control has required several different approaches, but the structural requirements of allostery are being defined.
The muscle enzyme is activated by both AMP and covalent phosphorylation, but of the two, covalent phosphorylation is dominant. Generally, the needs of the whole organism, however, supersede those of the individual cell. Thus, phosphorylases which are responsible for global glucose regulation, such as the yeast and mammalian liver
phosphorylase kinase. The phosphorylated state of the enzyme is controlled by extracellular signals, such as epinephrine (adrenaline), which act through the cAMP-dependent protein kinase cascade to turn on phosphorylase kinase. Activation by phosphorylation is reversed by the reciprocal action of protein phosphatase, PP12.
enzymes, are primarily activated by phosphorylation. In mammalian phosphorylases, Ser]4 is the substrate for
Ultimately, phosphorylase is controlled by glucose. When glucose is bound to the phosphorylated enzyme,
© 1992, Elsevier Science Publishers, (UK) 0376-5067/92/$05.00
TIBS 17
- FEBRUARY 1992
the complex becomes an excellent substrate for protein phosphatase. Once dephosphorylated, the enzyme with glucose bound is not
cap
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AMP. Glucose, therefore, can also control the AMPactivated, muscle enzyme, although it is thought to be more important in the controlofthephosphorylated, liver enzyme. Deprived of both the phosphate on Serl4 and AMP, the inactive phOsphorylase is again responsive to intra- and extracellular signals.
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phosphorylase is necessarily allosteric, because the enzyme is always firmly attached to its substrate in the glycogen particle. Degradation of glycogen by phosphorylase could pro!/¢~ y ceed until completion were it not for the opposing cel335- 332 3 6 1 - 370 lular signals. The sub¢z9 ¢~11 strates, GIc-I-P, inorganic phosphate and glycogen, Figure1 are allosteric effectors of Amino-terminaldomain of muscle phosphorylase. Schematic representation of the secondary structhe enzyme, but it is the two tural elements./he functionalsegments of the protein are named accordingto their function(gate, to activators (phosphorylated the active site; cap, of the activation locus; storage, site for glycogen)or their appearance (tower, Ser14 and AMP) and the above subunit; salute, over the glycogenstorage site; Dali, ~ribbon, draped across carboxy-terminal two inhibitors (Glc-6-P and domain). Most of these emanate from the carboxylterminiof the ~-sheets. glucose) that are responsible for the fine control of glycogenThe monomer can be divided into site of catalysis is located between the olysis in vivo 3. two large a/l[~ domains, the amino- and amino- and carboxy-terminal domains Many of the structural features carboxy-terminal domains (Figs I and of the monomer (Fig. 3). Proposals for responsible for the allosteric behavior 2). The amino-terminal domain (amino the catalytic mechanism based on of phosphorylase have recently been acids 1-482, Fig. 1) is one of the largest structures of substrate complexes have delineated. Specifically, we will focus on domains defined by X-ray crystal- been extensively discussedmL how information from several lines of lography. The dimer interface involves What structural features are responinvestigation is converging to provide a residues only in the amino-terminal sine for the allosteric responses of detailed understanding of one of domain. This domain also contains the phosphorylase to its various ligands nature's most elegant regulatory mech- activation locus, the site of Ser14 and substrates? This question can be anisms - the regulation of phosphoryl- phosphorylation and AMP binding, addressed in two parts: how does the ase by phosphorylation and AMP. More Topologically, this domain can be activated enzyme differ in structure coml~rehensive reviews are available 4-7. described as a nine-stranded, mostly from the inactive enzyme, and what parallel ~sheet core, surrounded by 16 allows the changes in one subunit of The phosphorylase molecule a-helices 8. The a-helices consist of the protein to be communicated to the Phosphorylase is a large protein approximately 200 amino acids and for other? Although the answers to these which can exist in many different struc- the most part, these helices connect questions are not yet obvious, an extural conformations, depending on the the 13-sheets of the protein core. amination of the functional elements of phosphorylation state of the enzyme Remarkably, all of the functional units the enzyme helps in identifying the and the presence or absence of various of the amino-terminal domain are con- structural units that are responsible for ligands. The phosphorylase monomer tained within the a-helical subset of the allosteric nature of the enzyme. from muscle contains 842 amino acid structural elements (named in Fig. 1). Subunitcontacts. The interactions beresidues (M=97434) and includes the The core of the carboxy-terminai do- tween the two monomers are arguably coenzyme pyridoxal phosphate, co- main has a dinucleotide binding fold9 the most important feature of the phosvalently attached to Lys680. The en- flanked by a-helical bundles (Fig. 2). phorylase dimer but these subunit conzyme is active as a dimer but may also The carboxy-terminal domain contains tacts involve only 200 of the nearly form an inactive tetramer, the pyridoxal phosphate cofactor. The 13 000 non-hydrogen atoms ]2. It is 67
TIBS 17 - FEBRUARY1992
date whichever ligand is available at the greatest relative chemical concentration, measuring the ambient energy surplus or deficiency. Of the three, only AMP activates the enzyme, properly juxtaposing the cap and helix 2 at an optimal distance with the greatest free energy Of interactiOnlT" Inhibitory locus. Access to the active site of phosphorylase is gained by disordering or moving the active site gate~6JL Stabilizing the gate from either
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through this relatively small number of atoms that the allosteric response is communicated. On the catalytic side of the dimer, subunit contacts are made by the tower helices (261-277), the car-
strengthening the subunit interactions, On the catalytic face, however, there are fewer subunit contacts when the enzyme is activated, Activationsite. AMP and phosphoserine~
boxyl termini of the Dali loops (184--185) and an interfacing ~-strand (193-195). On the other side, known as the regulatory face, contacts are made through (x-helices (the cap, 40-45, and helix 2, 48-78, in Figs 1 and 3). Subunit contacts change in number and identity depending on which of the various activators and inhibitors are bound to the enzyme]3-~. Less clear, is how the activation sites are linked to the catalytic sites and what links the two active sites to each other, Experiments show that when phosphorylase is activated, the contact area between subunits increases. A comparison of the structures of the phosphorylated enzyme and the dephosphorylated enzyme showed that the amino terminus (residues 5-16) is disordered in the dephosphorylated structure Is. When Serl4 is phosphorylated, Arg69 of the same monomer and Arg43 of the other monomer form strong ionic .bonds to the oxygens of the serine phosphate (Fig. 3, top inset). The amino terminus of the phosphorylated enzyme is bound to the body of the protein by polar and hydrophobic interactions, providing new contacts between monomers and 68
14 bind at non-overlapping sites within the subunit interface with their phosphate groups separated by over 16 A (Refs 14, 16). The activation locus is approximately 35 ]k from where the glycogen chain is bound within the active site (Fig. 3). Both AMP and phosphoserine are bound between the cap of one monomer and helix 2 of the other, which move towards each other, when the enzyme is activated. Phosphoserine and AMP form part of the subunit interrace, enhancing subunit contacts, although the particular atomic interactions are different for each ligand (Fig. 3, insets). The trigger which opens the active sites, admitting substrate, can be defined by what is common to these two activators, namely, the approach and interaction of the cap and helix 2. Whether this increased accessibility of the active site is caused by changes in subunit contacts or by changes in the positions of secondary structural elements within each subunit is not clear, The flexibility of the activation locus is remarkable in that Glc-6-P, ATP and AMP all bind using different subsets of amino acid side chains~4.1E The neighboring side chains move to accommo-
the inside or outside of the active site blocks this motion (Fig. 3, bottom inset). Inhibitors such as adenosine or caffeine shut the gate from the outside, by enhancing contacts between the two domains within a monomer. Glucose inhibits by shutting the gate from inside the active site. In addition to the hydrogen bonds formed with glucose, other contacts between the two domains are stabilized by the binding of glucose. The design of the gate easily shows how glucose and caffeine can bind simultaneously and synergistically inhibit activity [8. This direct connection between the inhibitory locus and the active site is deceptively simple; this locus is more complicated than we have described ~9. For example, the glucose-bound enzyme binds AMP very poorly.
Relatingstructural changes to function Sequence0fevents. As each new atomic structure of phosphorylase from different steps in the activation pathway is determined, we can assemble them into a kind of time-lapse series which describes the conformational changes of the enzyme. The complete series begins with the inactive, dephosphoryiated enzyme with inhibitors bound and ends with the phosphorylated enzyme complexed with substrates. In this series, we have snapshots of the dephosphorylated, inactive enzyme alone2°, and with inhibitor or substrate bound ~°,u, and in the presence of activating ligands ~3,~6.The series also includes structures of the phosphorylated enzyme complexed with glucose8, with the ligands AMP or ATP present ]4, or with substrate bound a. A view of the enzyme in a very active crystalline state was provided by the structure of the enzyme with AMP bound at the activation locus and with a co-enzyme annlog at the active site ~7.This analog miraics the presence of substrate in the transition state. From this series of structures, we can delineate the tertiary and quaternary movements which
TIBS 17 - FEBRUARY 1992
occur in response to activators, substrates and inhibitors. The tertiary and quaternary movemerits are a consequence of the rearrangement of secondary structural elements as a whole (Fig. 4). At the tertiary level, two major movements are seen; the central water cavity between the monomers contracts and the aminoand carboxy-terminal domains of a monomer move away from each other (compare the top structures in Fig. 4) 14. This movement of the domains bec°mes m°re dramatic as the m°lecule is more fully activated and can involve as much as a 5° rotation ~7. The quaternary motion is clam-like, with each half shell representing a monomer. Activation causes the clam shells to open more fully and twist slightly about the point of attachment (bottom cartoon, Fig. 4) 13-17. The magnitude of both the tertiary and quaternary movements differ in the various reported structures (M. F. Browner et al., submitted). In the inactive state, the gate holds the domains of the monomer together, controlling access to the site of catalysis. Upon activation these domains move apart and this is associated with the clam-like movement of the subunits, A central question in understanding the allosteric mechanism, however, remains: h o w are the structural changes in one monomer communicated to the other? One suggestion is that the tower helices
are essential in the clam-like opening, providing the link between the two active sites 13. In this view, the movement of the tower helices stabilizes the activated conformation. Another possibility is that the tower helices stabilize the inactive conformation, where the less flexible gate closes the active site to substrates. Our most recent snapshot of an activated structure shows that everything on either end of the tower helices is disordered ~7,leading us to ask if the tower helices play an active or passive role in opening the active sites, Phos~offlase homologs.The large database of primary phosphorylase sequences is another source of information of use in understanding allosteric regulation. Currently, sequence data are available for at least six mammalian isozymes22-25, two potato phosphorylases 26,27,two bacterial homologs~,29, two slime mold genes3°,31, and one yeast phosphorylase32. It is clear that all these homologs fall into onegenefamily6. These sequences provide information about the evolutionary develop-
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Rgure 3 The phosphorylasedimer and its functionalunits. The ct-carbontrace of the phosphorylated phosphorylasedimer looking at the regulatoryface. The twofold axis which relates the monomersis perpendicularto the page; rotatingthe molecule around an axis parallel to the page wouldexpose the catalyticface. The top inset shows the interactionsof phosphoserine14 of one monomerwith arginine 69 of the same monomerand 43 of the other. The middle inset is a close-up of the AMP binding site. The bottom inset shows the catalytic site from the perspective of the cofactor (the pyridoxal ring is perpendicular to the page), with glucose (GLC) bound in the active site and caffeine (CFF) bound on the outside of the gate. Amino acid residues are numbered; residues from the second monomer are repre-
sentedby adding1000 to the residue number. ment of allosteric regulation and how regulatory properties are tailored to meet tissue-specific needs. The mammalian enzymes are the most highly regulated, with a phosphorylation site and multiple allosteric sites. In contrast, the plant and bacterial phosphorylases are completely unregulated, Amino acids known to be important in binding the effector ligands are missing from the sequences of the prokaryotic and plant enzymes. A preliminary report of the atomic structure of potato phosphorylase suggests that its structure is more like that of the active, rather than the inactive, form of the muscle enzyme3a. This makes sense, since the potato enzyme is unregulated. The yeast enzyme, regulated by phosphorylation alone, falls midway between the mammalian and unregulated plant enzymes in the complexity of its regulation. We will soon know how the structure of the inactive yeast enzyme compares with other inactive phos-
phorylases since the X-ray structure of the enzyme from S. cerevisiae has recently been solved (V. L. Rath et al., unpublished). In contrast to their disparate regulatory properties, the kinetic behavior of the homologs is strikingly similar, because there is strict conservation of active site residues 34. Differences between tissue-specific isozymes provide yet another source of information about the intricacies of allosteric regulation. The mammalian phosphorylases are conserved between species but have diverged within a species by tissue type. Rabbit, rat and human muscle phosphorylase sequences are 97% identical, whereas human brain, muscle and liver phosphorylases are 80% identical in pairwise sequence comparisons23-2s,3s. These isozymes show distinct regulatory characteristics related to their different physiological roles 6. The tissue-specific differences are a result of modulation, rather than loss or gain, of allosteric
69
TIBS 17 - FEBRUARY 1 9 9 2
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Figure 4 Tertiary and quaternary structural changes which occur when phosphorylase is activated, The top of the figure shows space filling models of the co-carbon trace of inactive (left)s,15 and active phosphorylase dimers (right)17. Arrows and fiducial lines are identically positioned in the two diagrams and therefore provide references for the tertiary movements that occur when the enzyme is activated. For example, the lines at the top and bottom of the dimer are further from the carboxy-terminaldomain, which has moved in the direction of the arrow, awayfrom the amino-terminaldomain. Where the two monomers contact, the contacts are tightened in the active structure and the water cavity is compressed (represented by the lines and arrows between monomers). The clams at the bottom of the figure, which are in the same orientation as the dimers, dramatize the opening of the two monomers (quaternarymovements) upon activation,
this isozyme consumes the body's reserve energy supply at a moderate pace, due to its lower enzymatic activity when compared to the muscle enzyme. This natural modulation of liver activation is, in part, due to amino acid substitutions within the amino-terminal 48 residues. These substitutions are involved in contacts between the two monomers, but none are directly involved in binding AMP or phosphoserine. How subunit contacts modulate enzymatic activity is not yet clear. Subtle biochemical differences also distinguish the muscle and liver isozymes. The liver enzyme binds AMP non-cooperatively. The muscle enzyme, which must be responsive to acute demands for energy, binds this potent activator in a cooperative manner. A detailed modelling study39 leads to the suggestion that residue 48, which is different in the two isozymes, could play a critical role in linking the two AMP sites, since it connects the cap to helix 2. The symmetrically related cap and helix 2 change conformations when the enzyme is activated. The torsional rigidity of proline was thought to be essential in the correct positioning of these structural elements when AMP binds. Replacement of Pro48 in the muscle isozyme with either Thr (the
amino acid residue in liver), or Gly, abolished cooperative binding but the binding constant for AMP was unchanged (M. F. Browner et al., submitted). The requirement for proline between the cap and helix 2 is not absolute. Sub-
regulation. Our efforts to understand the tailoring of allosteric regulation have led us to develop a new set of biochemical tools for phosphorylase.
engineering new allosteric sites that duplicate the effect of the activation locus, Tinkering with allostety. Can different regulatory properties be assigned to The next chapter particular segments of the protein? One Protein engineering is a powerful experiment, in which eight of the first approach for analysing allosteric mech- 48 amino acids of the liver enzyme were anisms3E Based on abundant crystallo- substituted with the corresponding graphic data, domains and amino acids residues from muscle phosphorylase, can be assigned specific, testable roles suggested why the liver enzyme is less in promoting various conformational sensitive to activation than the muscle changes. Muscle and liver phosphoryl- isozyme37. The muscle-liver hybrid was use isozymes, as well as variants of activated by AMP and, surprisingly, each, have been produced and purified more active after phosphorylation than in milligram quantities from E. coli 37,38. the native liver enzyme. The results Three different approaches are taken in from this experiment highlight the trying to understand the details of the physiological differences between the allosteric mechanism: (1) construction muscle and liver enzymes, while pointof hybrids that swap domains from the ing us toward the structural features different isozymes to test hyp6theses which account for those differences, about the structural requirements of Liver phosphorylase is responsible for tissue specificity, (2) mutagenesis of maintaining whole body glucose levels single amino acids thought to have a and has been designed to respond only role in the ailosteric response, and (3) to extracellular signals. Once activated,
70
stitution of Ala had no effect on the cooperative binding of AMP. All variants were fully activated by phosphorylation. The Thr substitution was unique in that it was not fully activated by AMP. Crystallographic analysis at 2.7 ]~ }esolution of the Thr and Gly variants, in the absence of AMP, showed that replacement of residue 48 resulted in no significant structural changes. The fact that Pro48Thr is poorly activated by AMP, but fully activated by phosphorylation, suggests that the cooperativity in the AMP activation locus can be uncoupled, as nature has done in the liver enzyme. Re-inventingall0stery. In its simplest incarnation, the activation switch presumably requires only that the two monomers move toward each other, strengthening the contacts at the dimer interface either by AMP or phosphoserine binding. Can another mode of activation be added using the existing machinery and a new ligand-binding
TIBS 17 - FEBRUARY 1 9 9 2 site? The amino acid residues, Va145 and Tyr75, which are within van der Waals and hydrogen bonding distance of the adenine of AMP in the activated conformation, were replaced with histidine (M. F. Browner et al., unpublished). In the activated conformation, this pair of histidine side chains could coordinate a metal atom. This m u t a n t
phosphorylase
binds
zinc
chloride
tightly and cooperatively, and is thereby activated. Thus, a n e w allosteric site
for muscle phosphorylase was engineered. An atomic structure is needed to show that metal binding has recreated the conformational changes associated
with activation. If this is the case, then the placement of metal binding sites may be used as a sensitive probe to understand the extent and direction of m o v e m e n t required for the activation of phosphorylase. What have we learned about allosteric mechanisms? We c a n n o w clearly
identify the structural changes associated with the activation of phosphoryluse. It is, however, difficult at this point to say how these conformational changes are triggered. The dimer interface is clearly important and echoes a common theme in allosteric mechanisms 7. The structural elements which link the active sites remain a mystery, but one that we hope to solve using the approaches just described. Phosp h o r y l a s e can b e s t be u n d e r s t o o d as
Enzymes (Herv~, G. L., ed.), pp. 81-127, CRC Reviews 6 Newgard, C. B., Hwang, P. K. and Fletterick, R. J. (1989) Crit. Rev. Biochem. Mol. Biol. 24,
69-99
3850-3857
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Acknowledgements We thank Drs S. Sprang, E. Goldsmith and especially Virginia Ruth for their
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528-532 15 Sprang, s. R. et al. (1988) Nature 336, 216-221 16 Barford, D., Hu, S-H. and Johnson, L. N. (1991) J. Mol. Biol. 218, 233-260 17 Sprang, S. R. et al. (1991) Science 254, 1367-1371 18 Kasvinsky, P. J., Madsen, N. B., Sygusch, J. and Fletterick, R. J. (1978) J. Biol. Chem. 253, 9102-9106 19 Sprang, 8. R. et al. (1982) Biochemistry 21,
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(1991) in Protein-CarbohydrateInteractions,
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Transactions (Einspahr, H., ed.), pp. 427-434, American Crystallographic Association 22 Nakano, K., Hwang, P. K. and Fletterick, R.J. (1986) FEBS Lett. 204, 283-287 23 Newgard, C. B., Nakano, K., Hwang, P. K. and
a
biological transducer, taking cues from its environment, integrating the signals, and responding by adjusting its structural conformation and activity level.
Fletterick, R. J. (1986) Prec. Natl Acad. Sci. USA 83, 8132-8136 24 Newgard, C. B., Littman, D. R., van Genderen, C. and Fletterick, R. J. (1988) J. Biol. Chem. 263,
38 Browner, M. F., Rasor, P., Tugendreich, S. and Fletterick, R. J. (1991) Protein Eng. 4, 351-357 39 Rath, V. L., Newgard, C. B., Sprang, G. E. J. and Fletterick, R. J. (1987) Proteins: Struct. Funct. Genet. 2, 225-235
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References
~
4812-4817 3 Hers, H. G. (1990)J. InheritedMetab. Dis. 13, 395-410 4Johnson, L.N.(1989) CaflsbergRes. Commun.
54, 203-229
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critical readingcomments,Of theandearlYJuliedraftSNewdolland ~::: '::: ..... ~:: : : thoughtful for her artistic interpretation of the quaternary movement of phosphoryl~,~ ase. The work from the laboratory of ...... ~.: R. J."T. was supported by National ~ ' ~ : ~ i ~ :: ..... ::::,:i::: Institutes of Health (DK26082-12 and DK32822-07). M. F. B. was supported by ::: : a National Institutes of Health postdoc:~,l$t¢~G~, :::: : : i : , . . . . toral fellowship(GM12201). ~ . : ::.: :::i. : ~:: :, : i : : :
1 Fletterick, R. J. and Madsen, N. B. (1980)Annu. ev. Biochem. 49, 31-61 2 Gratecos, D., Detwiler, T. C., Hurd, S. and Fischer, E. H. (1977) Biochemistry 16,
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Illinois, PO Box 6998, Chicago; iL 60680, USA.
5 Johnson, L. N. et al. (1989) in AIIostedc
71
Council and The G6ran Gustafsson Foundation for financial support. We wish to thank our many colleagues who have worked with us o n various properties of photosystem II and its photoinactivation and in particular, Steven
Hall and Hugh Salter for helping us prepare the figures used in this article.
References 1 Kyle, D. J., Osmond, C. B. and Arntzen, C.J. (1987) Photoinhibition, Topics in Photosynthesis (Barber, J., ed.) (Vol. 9), Elsevier 20smond, C. B. and Chow, W. S. (1988) Aust. J. Plant PhysioL 15, 1-9 3 Ewart, A. J. (1896) J. Linn. Soc. 31, 364-461 4 Andersson, B. and Styring, S. (1991) in Current Topics in Bioenergetics (Lee, C. P., ed.) (Vol. 16), pp. 1-81, Academic Press 5 Anderson, J. M. and Andersson, B. (1988) Trends Biochem. Sci. 13, 351-355 6 Kyle, D. J., Ohad, I. and Amtzen, C. J. (1984) Proc. Natl Acad. Sci. USA 81, 4070-4071 7 Mattoo, A. K., Marder, J. B. and Edelman, M. (1989) Cell 56, 241-246 8 Michel, H. and Deisenhofer, J. (1988) Biochemistry 27, 1-7 9 Trebst, A. (1986) Z. Naturforsch. 41c, 240-245 10 Barber, J. (1987) Trends Biochem. Sci. 12, 321-326 11 Rutherford, A. W. (1989) Trends Biochem. Sci.
GLYCOGEN PHOSPHORYLASE is an essential enzyme which, in mammals, provides the fuel needed to sustain life between feeding. Phosphorylase senses the metabolic state of the cell or organism, and responds by liberating glucose from stored glycogen as needed. Phosphorylase is studied both for its central role in an important biological process and also as a model for allosteric behavior in proteins, The activation and inhibition of phosphorylases are ceil, tissue and organism specific. In mammals, phosphorylase must be switched from its dormant state to the active form in order to liberate glucose 1-phosphate (Glc-I-P) from glycogen, with the help of inorganic phosphate 1. Inside muscle cells, an increase in the products of ATP hydrolysis, principally AMP, signals the enzyme to commence digesting glycogen. Activation of phosphorylase by AMP can be competitively inhibited by metabolic indicators of ample energy, such as glucose 6-phosphate.(Glc-6-P) and ATP. M. F. Browner and R. J. Fletterick are at the Department of Biochemistry and Biophysics, Box 0448, Universityof California, San Francisco, CA 94143-0448, USA.
66
14,227-232
Acta1060,1-12
12 Prasil, 0., Adir, N. and Ohad, I. (1992) in The Photosystems, Topics in Photosynthesis
(Barber, J., ed.) (Vol. 11), pp. 220-250, Elsevier 13 Eaglesham, A. R. J. and Ellis, R. J. (1974) Biochim.Biophys. Acta 335, 396-407 14 Edelman, M. and Reisfeld, A. (1978) in Chloroplast Development (Akoyunoglou,G. and
Argyroudi-Akoyunoglou,J. H., eds), pp. 641-852,
Elsevier
27 Schuster, G., Timberg, R. and Ohad, I. (1988) Eur. J. Biochem. 177, 403-410 28 Trebst, A., Depka, B. and Kipper, M. (1990) in Current Research in Photosynthesis
(Baltscheffsky, M., ed.) (Vol. 1), pp. 217-222, Kluwer Academic 29 Aro, E-M., Hundal, T., Carlberg, I. and Andersson, B. (1990) Biochim. Biophys. Acta
1019, 269-275
15 Greenberg, B. M. et al. (1989) Proc. Natl Acad. Sci. USA 86, 6617-8620 16 Strid, A., Chow, W. S. and Anderson, J.M. (1990) Biochim. Biophys. Acta 1020,
260-268
30 Virgin, I., Ghanotakis, D. F. and Andersson, B. (1990) FEBSLett. 269, 45-48 31 Virgin, I., Salter, H., Ghanotakis, D. F. and Andersson, B. (1991) FEBSLett. 287,
125-128
17 Setlik, I. et al. (1990) Photosynth. Res. 23,
39-48 18 Vass, I. et al. Proc. NatlAcad. Sci. USA (in
press) 19 Telfer, A., He, W-Z. and Barber, J. (1990) Biochim. Biophys. Acta 1017, 143-151 20 Shipton, C. A. and Barber, J. (1991) Proc. Natl Acad. Sci. USA 88, 6691-6695 21 Hundal, T., Aro, E. M., Carlberg, I. and Andersson, B. (1990) FEBS Lett. 267, 203-206 22 Kirilovsky, D. and Etienne, A-L. (1991) FEBS Lett. 279, 201-204 23 Telfer, A., De Las Rivas, J. and Barber, J. (1991) Biochim. Biophys. Acta 1060, 106-114 24 Callahan, F. E., Becker, D. W. and Cheniae, G.M. (1986) Plant Physiol. 82, 261-269 25 Jegerschold, C. and Styring, S. (1991) F E B S Lett. 280, 87-90 26 Mayes, S. R. et al. (1991) Biochim. Biophys.
32 Shipton, C. A. and Barber, J. Biochim. Biophys. Acta (in press) 33 Greenberg, B. M., Gaba, V., Mattoo, A. K. and Edelman, M. (1987) EMBOJ. 6, 2865-2869 34 Trebst, A. and Depka, B. (1990) Z. Naturforsch.
45c, 765-771 35 Barbato, R., Shipton, C. A., Giacometti, G. M. and Barber, J. (1991) FEBSLett. 290, 162-166 36 Krause, H. G. and Behrend, U. (1986) FEBS Lett. 200, 298-302 37 Demmig-Adam,B. (1990) Biochim. Biophys. Acta 1020, 1-24 38 Takahashi, Y., Hansson, 6 , Mathis, P. and Satoh, K. (1987) Biochim. Biophys. Acta 893,
49-59 39 Thompson, L. K. and Brudvig, G. W. (1988) Biochemistry 27, 6653-6658 40 Somersalo, S. and Krause, G. H. (1989) Planta
177, 409-416
Phosphorylase: a biological transducer
A transducer is a device that receives energy from one system and transmits it, often in a different form, to another. Glycogen phosphorylase receives information from the cell or organism in the form of metabolic signals. The energy associated with the binding of these ligand signals is integrated and transmitted at an atomic level, allowing precise adjustment of the enzymatic activity. Understanding this elegant allosteric control has required several different approaches, but the structural requirements of allostery are being defined.
The muscle enzyme is activated by both AMP and covalent phosphorylation, but of the two, covalent phosphorylation is dominant. Generally, the needs of the whole organism, however, supersede those of the individual cell. Thus, phosphorylases which are responsible for global glucose regulation, such as the yeast and mammalian liver
phosphorylase kinase. The phosphorylated state of the enzyme is controlled by extracellular signals, such as epinephrine (adrenaline), which act through the cAMP-dependent protein kinase cascade to turn on phosphorylase kinase. Activation by phosphorylation is reversed by the reciprocal action of protein phosphatase, PP12.
enzymes, are primarily activated by phosphorylation. In mammalian phosphorylases, Ser]4 is the substrate for
Ultimately, phosphorylase is controlled by glucose. When glucose is bound to the phosphorylated enzyme,
© 1992, Elsevier Science Publishers, (UK) 0376-5067/92/$05.00
TIBS 17
- FEBRUARY 1992
the complex becomes an excellent substrate for protein phosphatase. Once dephosphorylated, the enzyme with glucose bound is not
cap
sens,t,veto by t°wer " I
AMP. Glucose, therefore, can also control the AMPactivated, muscle enzyme, although it is thought to be more important in the controlofthephosphorylated, liver enzyme. Deprived of both the phosphate on Serl4 and AMP, the inactive phOsphorylase is again responsive to intra- and extracellular signals.
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phosphorylase is necessarily allosteric, because the enzyme is always firmly attached to its substrate in the glycogen particle. Degradation of glycogen by phosphorylase could pro!/¢~ y ceed until completion were it not for the opposing cel335- 332 3 6 1 - 370 lular signals. The sub¢z9 ¢~11 strates, GIc-I-P, inorganic phosphate and glycogen, Figure1 are allosteric effectors of Amino-terminaldomain of muscle phosphorylase. Schematic representation of the secondary structhe enzyme, but it is the two tural elements./he functionalsegments of the protein are named accordingto their function(gate, to activators (phosphorylated the active site; cap, of the activation locus; storage, site for glycogen)or their appearance (tower, Ser14 and AMP) and the above subunit; salute, over the glycogenstorage site; Dali, ~ribbon, draped across carboxy-terminal two inhibitors (Glc-6-P and domain). Most of these emanate from the carboxylterminiof the ~-sheets. glucose) that are responsible for the fine control of glycogenThe monomer can be divided into site of catalysis is located between the olysis in vivo 3. two large a/l[~ domains, the amino- and amino- and carboxy-terminal domains Many of the structural features carboxy-terminal domains (Figs I and of the monomer (Fig. 3). Proposals for responsible for the allosteric behavior 2). The amino-terminal domain (amino the catalytic mechanism based on of phosphorylase have recently been acids 1-482, Fig. 1) is one of the largest structures of substrate complexes have delineated. Specifically, we will focus on domains defined by X-ray crystal- been extensively discussedmL how information from several lines of lography. The dimer interface involves What structural features are responinvestigation is converging to provide a residues only in the amino-terminal sine for the allosteric responses of detailed understanding of one of domain. This domain also contains the phosphorylase to its various ligands nature's most elegant regulatory mech- activation locus, the site of Ser14 and substrates? This question can be anisms - the regulation of phosphoryl- phosphorylation and AMP binding, addressed in two parts: how does the ase by phosphorylation and AMP. More Topologically, this domain can be activated enzyme differ in structure coml~rehensive reviews are available 4-7. described as a nine-stranded, mostly from the inactive enzyme, and what parallel ~sheet core, surrounded by 16 allows the changes in one subunit of The phosphorylase molecule a-helices 8. The a-helices consist of the protein to be communicated to the Phosphorylase is a large protein approximately 200 amino acids and for other? Although the answers to these which can exist in many different struc- the most part, these helices connect questions are not yet obvious, an extural conformations, depending on the the 13-sheets of the protein core. amination of the functional elements of phosphorylation state of the enzyme Remarkably, all of the functional units the enzyme helps in identifying the and the presence or absence of various of the amino-terminal domain are con- structural units that are responsible for ligands. The phosphorylase monomer tained within the a-helical subset of the allosteric nature of the enzyme. from muscle contains 842 amino acid structural elements (named in Fig. 1). Subunitcontacts. The interactions beresidues (M=97434) and includes the The core of the carboxy-terminai do- tween the two monomers are arguably coenzyme pyridoxal phosphate, co- main has a dinucleotide binding fold9 the most important feature of the phosvalently attached to Lys680. The en- flanked by a-helical bundles (Fig. 2). phorylase dimer but these subunit conzyme is active as a dimer but may also The carboxy-terminal domain contains tacts involve only 200 of the nearly form an inactive tetramer, the pyridoxal phosphate cofactor. The 13 000 non-hydrogen atoms ]2. It is 67
TIBS 17 - FEBRUARY1992
date whichever ligand is available at the greatest relative chemical concentration, measuring the ambient energy surplus or deficiency. Of the three, only AMP activates the enzyme, properly juxtaposing the cap and helix 2 at an optimal distance with the greatest free energy Of interactiOnlT" Inhibitory locus. Access to the active site of phosphorylase is gained by disordering or moving the active site gate~6JL Stabilizing the gate from either
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Carboxy-terminal domain of muscle phosphorylase.The dinucleotide fold (560-714) is flanked by additional (~-helices.The pyridoxal phosphate cofactor is covalently bound to Lys680 in ~-25.
through this relatively small number of atoms that the allosteric response is communicated. On the catalytic side of the dimer, subunit contacts are made by the tower helices (261-277), the car-
strengthening the subunit interactions, On the catalytic face, however, there are fewer subunit contacts when the enzyme is activated, Activationsite. AMP and phosphoserine~
boxyl termini of the Dali loops (184--185) and an interfacing ~-strand (193-195). On the other side, known as the regulatory face, contacts are made through (x-helices (the cap, 40-45, and helix 2, 48-78, in Figs 1 and 3). Subunit contacts change in number and identity depending on which of the various activators and inhibitors are bound to the enzyme]3-~. Less clear, is how the activation sites are linked to the catalytic sites and what links the two active sites to each other, Experiments show that when phosphorylase is activated, the contact area between subunits increases. A comparison of the structures of the phosphorylated enzyme and the dephosphorylated enzyme showed that the amino terminus (residues 5-16) is disordered in the dephosphorylated structure Is. When Serl4 is phosphorylated, Arg69 of the same monomer and Arg43 of the other monomer form strong ionic .bonds to the oxygens of the serine phosphate (Fig. 3, top inset). The amino terminus of the phosphorylated enzyme is bound to the body of the protein by polar and hydrophobic interactions, providing new contacts between monomers and 68
14 bind at non-overlapping sites within the subunit interface with their phosphate groups separated by over 16 A (Refs 14, 16). The activation locus is approximately 35 ]k from where the glycogen chain is bound within the active site (Fig. 3). Both AMP and phosphoserine are bound between the cap of one monomer and helix 2 of the other, which move towards each other, when the enzyme is activated. Phosphoserine and AMP form part of the subunit interrace, enhancing subunit contacts, although the particular atomic interactions are different for each ligand (Fig. 3, insets). The trigger which opens the active sites, admitting substrate, can be defined by what is common to these two activators, namely, the approach and interaction of the cap and helix 2. Whether this increased accessibility of the active site is caused by changes in subunit contacts or by changes in the positions of secondary structural elements within each subunit is not clear, The flexibility of the activation locus is remarkable in that Glc-6-P, ATP and AMP all bind using different subsets of amino acid side chains~4.1E The neighboring side chains move to accommo-
the inside or outside of the active site blocks this motion (Fig. 3, bottom inset). Inhibitors such as adenosine or caffeine shut the gate from the outside, by enhancing contacts between the two domains within a monomer. Glucose inhibits by shutting the gate from inside the active site. In addition to the hydrogen bonds formed with glucose, other contacts between the two domains are stabilized by the binding of glucose. The design of the gate easily shows how glucose and caffeine can bind simultaneously and synergistically inhibit activity [8. This direct connection between the inhibitory locus and the active site is deceptively simple; this locus is more complicated than we have described ~9. For example, the glucose-bound enzyme binds AMP very poorly.
Relatingstructural changes to function Sequence0fevents. As each new atomic structure of phosphorylase from different steps in the activation pathway is determined, we can assemble them into a kind of time-lapse series which describes the conformational changes of the enzyme. The complete series begins with the inactive, dephosphoryiated enzyme with inhibitors bound and ends with the phosphorylated enzyme complexed with substrates. In this series, we have snapshots of the dephosphorylated, inactive enzyme alone2°, and with inhibitor or substrate bound ~°,u, and in the presence of activating ligands ~3,~6.The series also includes structures of the phosphorylated enzyme complexed with glucose8, with the ligands AMP or ATP present ]4, or with substrate bound a. A view of the enzyme in a very active crystalline state was provided by the structure of the enzyme with AMP bound at the activation locus and with a co-enzyme annlog at the active site ~7.This analog miraics the presence of substrate in the transition state. From this series of structures, we can delineate the tertiary and quaternary movements which
TIBS 17 - FEBRUARY 1992
occur in response to activators, substrates and inhibitors. The tertiary and quaternary movemerits are a consequence of the rearrangement of secondary structural elements as a whole (Fig. 4). At the tertiary level, two major movements are seen; the central water cavity between the monomers contracts and the aminoand carboxy-terminal domains of a monomer move away from each other (compare the top structures in Fig. 4) 14. This movement of the domains bec°mes m°re dramatic as the m°lecule is more fully activated and can involve as much as a 5° rotation ~7. The quaternary motion is clam-like, with each half shell representing a monomer. Activation causes the clam shells to open more fully and twist slightly about the point of attachment (bottom cartoon, Fig. 4) 13-17. The magnitude of both the tertiary and quaternary movements differ in the various reported structures (M. F. Browner et al., submitted). In the inactive state, the gate holds the domains of the monomer together, controlling access to the site of catalysis. Upon activation these domains move apart and this is associated with the clam-like movement of the subunits, A central question in understanding the allosteric mechanism, however, remains: h o w are the structural changes in one monomer communicated to the other? One suggestion is that the tower helices
are essential in the clam-like opening, providing the link between the two active sites 13. In this view, the movement of the tower helices stabilizes the activated conformation. Another possibility is that the tower helices stabilize the inactive conformation, where the less flexible gate closes the active site to substrates. Our most recent snapshot of an activated structure shows that everything on either end of the tower helices is disordered ~7,leading us to ask if the tower helices play an active or passive role in opening the active sites, Phos~offlase homologs.The large database of primary phosphorylase sequences is another source of information of use in understanding allosteric regulation. Currently, sequence data are available for at least six mammalian isozymes22-25, two potato phosphorylases 26,27,two bacterial homologs~,29, two slime mold genes3°,31, and one yeast phosphorylase32. It is clear that all these homologs fall into onegenefamily6. These sequences provide information about the evolutionary develop-
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Rgure 3 The phosphorylasedimer and its functionalunits. The ct-carbontrace of the phosphorylated phosphorylasedimer looking at the regulatoryface. The twofold axis which relates the monomersis perpendicularto the page; rotatingthe molecule around an axis parallel to the page wouldexpose the catalyticface. The top inset shows the interactionsof phosphoserine14 of one monomerwith arginine 69 of the same monomerand 43 of the other. The middle inset is a close-up of the AMP binding site. The bottom inset shows the catalytic site from the perspective of the cofactor (the pyridoxal ring is perpendicular to the page), with glucose (GLC) bound in the active site and caffeine (CFF) bound on the outside of the gate. Amino acid residues are numbered; residues from the second monomer are repre-
sentedby adding1000 to the residue number. ment of allosteric regulation and how regulatory properties are tailored to meet tissue-specific needs. The mammalian enzymes are the most highly regulated, with a phosphorylation site and multiple allosteric sites. In contrast, the plant and bacterial phosphorylases are completely unregulated, Amino acids known to be important in binding the effector ligands are missing from the sequences of the prokaryotic and plant enzymes. A preliminary report of the atomic structure of potato phosphorylase suggests that its structure is more like that of the active, rather than the inactive, form of the muscle enzyme3a. This makes sense, since the potato enzyme is unregulated. The yeast enzyme, regulated by phosphorylation alone, falls midway between the mammalian and unregulated plant enzymes in the complexity of its regulation. We will soon know how the structure of the inactive yeast enzyme compares with other inactive phos-
phorylases since the X-ray structure of the enzyme from S. cerevisiae has recently been solved (V. L. Rath et al., unpublished). In contrast to their disparate regulatory properties, the kinetic behavior of the homologs is strikingly similar, because there is strict conservation of active site residues 34. Differences between tissue-specific isozymes provide yet another source of information about the intricacies of allosteric regulation. The mammalian phosphorylases are conserved between species but have diverged within a species by tissue type. Rabbit, rat and human muscle phosphorylase sequences are 97% identical, whereas human brain, muscle and liver phosphorylases are 80% identical in pairwise sequence comparisons23-2s,3s. These isozymes show distinct regulatory characteristics related to their different physiological roles 6. The tissue-specific differences are a result of modulation, rather than loss or gain, of allosteric
69
TIBS 17 - FEBRUARY 1 9 9 2
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q
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Figure 4 Tertiary and quaternary structural changes which occur when phosphorylase is activated, The top of the figure shows space filling models of the co-carbon trace of inactive (left)s,15 and active phosphorylase dimers (right)17. Arrows and fiducial lines are identically positioned in the two diagrams and therefore provide references for the tertiary movements that occur when the enzyme is activated. For example, the lines at the top and bottom of the dimer are further from the carboxy-terminaldomain, which has moved in the direction of the arrow, awayfrom the amino-terminaldomain. Where the two monomers contact, the contacts are tightened in the active structure and the water cavity is compressed (represented by the lines and arrows between monomers). The clams at the bottom of the figure, which are in the same orientation as the dimers, dramatize the opening of the two monomers (quaternarymovements) upon activation,
this isozyme consumes the body's reserve energy supply at a moderate pace, due to its lower enzymatic activity when compared to the muscle enzyme. This natural modulation of liver activation is, in part, due to amino acid substitutions within the amino-terminal 48 residues. These substitutions are involved in contacts between the two monomers, but none are directly involved in binding AMP or phosphoserine. How subunit contacts modulate enzymatic activity is not yet clear. Subtle biochemical differences also distinguish the muscle and liver isozymes. The liver enzyme binds AMP non-cooperatively. The muscle enzyme, which must be responsive to acute demands for energy, binds this potent activator in a cooperative manner. A detailed modelling study39 leads to the suggestion that residue 48, which is different in the two isozymes, could play a critical role in linking the two AMP sites, since it connects the cap to helix 2. The symmetrically related cap and helix 2 change conformations when the enzyme is activated. The torsional rigidity of proline was thought to be essential in the correct positioning of these structural elements when AMP binds. Replacement of Pro48 in the muscle isozyme with either Thr (the
amino acid residue in liver), or Gly, abolished cooperative binding but the binding constant for AMP was unchanged (M. F. Browner et al., submitted). The requirement for proline between the cap and helix 2 is not absolute. Sub-
regulation. Our efforts to understand the tailoring of allosteric regulation have led us to develop a new set of biochemical tools for phosphorylase.
engineering new allosteric sites that duplicate the effect of the activation locus, Tinkering with allostety. Can different regulatory properties be assigned to The next chapter particular segments of the protein? One Protein engineering is a powerful experiment, in which eight of the first approach for analysing allosteric mech- 48 amino acids of the liver enzyme were anisms3E Based on abundant crystallo- substituted with the corresponding graphic data, domains and amino acids residues from muscle phosphorylase, can be assigned specific, testable roles suggested why the liver enzyme is less in promoting various conformational sensitive to activation than the muscle changes. Muscle and liver phosphoryl- isozyme37. The muscle-liver hybrid was use isozymes, as well as variants of activated by AMP and, surprisingly, each, have been produced and purified more active after phosphorylation than in milligram quantities from E. coli 37,38. the native liver enzyme. The results Three different approaches are taken in from this experiment highlight the trying to understand the details of the physiological differences between the allosteric mechanism: (1) construction muscle and liver enzymes, while pointof hybrids that swap domains from the ing us toward the structural features different isozymes to test hyp6theses which account for those differences, about the structural requirements of Liver phosphorylase is responsible for tissue specificity, (2) mutagenesis of maintaining whole body glucose levels single amino acids thought to have a and has been designed to respond only role in the ailosteric response, and (3) to extracellular signals. Once activated,
70
stitution of Ala had no effect on the cooperative binding of AMP. All variants were fully activated by phosphorylation. The Thr substitution was unique in that it was not fully activated by AMP. Crystallographic analysis at 2.7 ]~ }esolution of the Thr and Gly variants, in the absence of AMP, showed that replacement of residue 48 resulted in no significant structural changes. The fact that Pro48Thr is poorly activated by AMP, but fully activated by phosphorylation, suggests that the cooperativity in the AMP activation locus can be uncoupled, as nature has done in the liver enzyme. Re-inventingall0stery. In its simplest incarnation, the activation switch presumably requires only that the two monomers move toward each other, strengthening the contacts at the dimer interface either by AMP or phosphoserine binding. Can another mode of activation be added using the existing machinery and a new ligand-binding
TIBS 17 - FEBRUARY 1 9 9 2 site? The amino acid residues, Va145 and Tyr75, which are within van der Waals and hydrogen bonding distance of the adenine of AMP in the activated conformation, were replaced with histidine (M. F. Browner et al., unpublished). In the activated conformation, this pair of histidine side chains could coordinate a metal atom. This m u t a n t
phosphorylase
binds
zinc
chloride
tightly and cooperatively, and is thereby activated. Thus, a n e w allosteric site
for muscle phosphorylase was engineered. An atomic structure is needed to show that metal binding has recreated the conformational changes associated
with activation. If this is the case, then the placement of metal binding sites may be used as a sensitive probe to understand the extent and direction of m o v e m e n t required for the activation of phosphorylase. What have we learned about allosteric mechanisms? We c a n n o w clearly
identify the structural changes associated with the activation of phosphoryluse. It is, however, difficult at this point to say how these conformational changes are triggered. The dimer interface is clearly important and echoes a common theme in allosteric mechanisms 7. The structural elements which link the active sites remain a mystery, but one that we hope to solve using the approaches just described. Phosp h o r y l a s e can b e s t be u n d e r s t o o d as
Enzymes (Herv~, G. L., ed.), pp. 81-127, CRC Reviews 6 Newgard, C. B., Hwang, P. K. and Fletterick, R. J. (1989) Crit. Rev. Biochem. Mol. Biol. 24,
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References
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4812-4817 3 Hers, H. G. (1990)J. InheritedMetab. Dis. 13, 395-410 4Johnson, L.N.(1989) CaflsbergRes. Commun.
54, 203-229
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critical readingcomments,Of theandearlYJuliedraftSNewdolland ~::: '::: ..... ~:: : : thoughtful for her artistic interpretation of the quaternary movement of phosphoryl~,~ ase. The work from the laboratory of ...... ~.: R. J."T. was supported by National ~ ' ~ : ~ i ~ :: ..... ::::,:i::: Institutes of Health (DK26082-12 and DK32822-07). M. F. B. was supported by ::: : a National Institutes of Health postdoc:~,l$t¢~G~, :::: : : i : , . . . . toral fellowship(GM12201). ~ . : ::.: :::i. : ~:: :, : i : : :
1 Fletterick, R. J. and Madsen, N. B. (1980)Annu. ev. Biochem. 49, 31-61 2 Gratecos, D., Detwiler, T. C., Hurd, S. and Fischer, E. H. (1977) Biochemistry 16,
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