Structural and functional properties of Drosophila melanogaster phosphorylase: comparison with the rabbit skeletal muscle enzyme

Glycogen phosphorylase isolated from Drosophila melanogaster contains one pyridoxal 5'-phosphate per subunit; the coenzyme is in a hydrophobic environment. Fruit-fly phosphorylase a has lower KM for glucose-1-phosphate and is less sensitive to allosteric inhibitors than the b form of the enzyme. The amino acid composition of Drosophila phosphorylase differs from that of rabbit skeletal muscle phosphorylase. These two enzymes give distinct one dimensional peptide maps. The distribution of reactive SH-groups is markedly different in the insect and vertebrate phosphorylase. Fruit-fly phosphorylase a is dephosphorylated by either rabbit or Drosophila protein phosphatase-1 at a slower rate than rabbit muscle phosphorylase a.     

Mechanism of the phosphorylase reaction. Utilization of D-gluco-hept-1-enitol in the absence of primer

alpha-Glucan phosphorylases from rabbit skeletal muscle, potato tubers and Escherichia coli catalyze the utilization of 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (heptenitol) in the presence of arsenate or phosphate. 1H-NMR analysis in the presence of 2H2O and arsenate indicated formation of 1-[1-2H]deoxy-alpha-D-glucoheptulose with rates comparable to the arsenolysis of poly- or oligosaccharides. The reaction depends on the presence of a dianionic 5'-phosphate group of pyridoxal in the active conformation of the phosphorylases. Heptenitol is the first known substrate of alpha-glucan phosphorylases which does not require a primer. This is explained by the finding that heptenitol is exclusively used as substrate for the degradative pathway of the phosphorylase reaction where it competes with polysaccharide substrates. In the presence of phosphate the reaction product is 1-deoxy-alpha-D-gluco-heptulose 2-phosphate (heptulose-2-P), which subsequently inhibits the reaction. This characterizes heptulose-2-P as an enzyme-derived inhibitor. The Ki = 1.9 X 10(-6) M with potato phosphorylase suggests the formation of a transition-state-like enzyme-ligand complex. These findings, together with the fact that the phosphates of heptulose-2-P and pyridoxal 5'-phosphate are linked by hydrogen bridges [Klein, H. W., Im, M. J., Palm, D. & Helmreich, E. J. M. (1984) Biochemistry 23, 5853-5861], make it likely that both phosphates are involved in phosphorylase catalysis. A catalytic mechanism of phosphorylase action is proposed in which a 'mobile' phosphate anion plays a versatile role. It serves as proton carrier for the substrate activation, it stabilizes the intermediate and acts as a nucleophile which can accept a glycosyl residue reversibly.     

Potato and rabbit muscle phosphorylases: comparative studies on the structure,function and regulation of regulatory and nonregulatory enzymes

Summary Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.     

Dissociation constants of glucan phosphorylases of rabbit tissues studied by polyacrylamide gel disc electrophoresis

Using polyacrylamide gel disc electrophoresis, a simple and sensitive stain method for glucan phosphorylase (EC 2.4.1.1) was developed. With this method 0.3–1.5 μg or 1–5 units of phosphorylase could be demonstrated as a sharp band within a few hours. Mobility of phosphorylase fraction was retarded in gels containing glycogen. From the change of mobility as a function of glycogen concentrations, the dissociation constants of phosphorylases of rabbit skeletal muscle, liver, and brain with rabbit liver glycogen was calculated. They were 6.1 × 10⁻⁴, 22 × 10⁻⁴, and 13 × 10⁻⁴m, respectively. From the electrophoretic mobility, rabbit tissue phosphorylases could be classified into two: those of brain and kidney, and those of skeletal muscle and liver. When the electrophoresis gel, however, contained glycogen in a considerable concentration, their mobilities were retarded, and the retardation was more marked with those of skeletal muscle and brain than with those of liver and kidney. Hence, all four tissue phosphorylases could be distinguished only by the disc gel containing glycogen.     

Modeling the biochemical differences between rabbit muscle and human liver phosphorylase

Glycogen phosphorylases catalyze the regulated breakdown of glycogen to glucose-1-phosphate. In mammals, glycogen phosphorylase occurs in three different isozymes called liver, muscle, and brain after the tissues in which they are preferentially expressed. The muscle isozyme binds and is activated cooperatively by AMP. In contrast, the liver enzyme binds AMP noncooperatively and is poorly activated. The amino acid sequence of human liver phosphorylase is 80% identical with rabbit muscle phosphorylase, and those residues which contact AMP are conserved. Using computer graphics software, we replaced side chains of the known rabbit muscle structure with those of human liver phosphorylase and interpreted the effects of these changes in order to account for the biochemical differences between them. We have identified two substitutions in liver phosphorylase potentially important in altering the cooperative binding and activation of this isozyme by AMP.     

Dissociative mechanism of thermal denaturation of rabbit skeletal muscle glycogen phosphorylase b

The thermal stability of rabbit skeletal muscle glycogen phosphorylase b was characterized using enzymological inactivation studies, differential scanning calorimetry, and analytical ultracentrifugation. The results suggest that denaturation proceeds by the dissociative mechanism, i.e., it includes the step of reversible dissociation of the active dimer into inactive monomers and the following step of irreversible denaturation of the monomer. It was shown that glucose 1-phosphate (substrate), glucose (competitive inhibitor), AMP (allosteric activator), FMN, and glucose 6-phosphate (allosteric inhibitors) had a protective effect. Calorimetric study demonstrates that the cofactor of glycogen phosphorylase-pyridoxal 5'-phosphate-stabilizes the enzyme molecule. Partial reactivation of glycogen phosphorylase b preheated at 53 degrees C occurs after cooling of the enzyme solution to 30 degrees C. The fact that the rate of reactivation decreases with dilution of the enzyme solution indicates association of inactive monomers into active dimers during renaturation. The allosteric inhibitor FMN enhances the rate of phosphorylase b reactivation.     

Studies on phosphorylase isozymes in lower vertebrates: evidence for the presence of two isozymes in elasmobranchs.

Two distinct phosphorylase isozymes, skeletal muscle phosphorylase b and liver phosphorylase b, have been purified from skate (Raja pulchra) in a homogeneous form as judged by electrophoretic and immunological criteria. Both isozymes were dependent on AMP for activity and converted to a forms by rabbit muscle phosphorylase kinase. Their subunit molecular weight determined by sodium dodecyl sulfate-gel electrophoresis was 94,000. These isozymes were distinctly different in affinities for glycogen and AMP, while they were very similar in sensitivities to SO₄^2?. Rabbit antibodies against each of the muscle and liver isozymes inhibited completely the respective specific antigens. No cross-reaction was observed in double diffusion tests, but some immunological relatedness of these isozymes was demonstrated by inhibition tests with antibodies. Their similarity was also shown by amino acid analyses. No evidence has been obtained that the skate possesses such an isozyme as mammalian phosphorylase L, the b form of which is inactive even in the presence of AMP. Electrophoretic studies on phosphorylases of crucian carp, toad, and snake revealed that these animals possess three isozymes which strikingly resemble mammalian isozymes in the organ-specific distribution and electrophoretic behavior.     

Dephosphorylation and inactivation of phosphorylase kinase: subunit specificity of rabbit skeletal muscle protein phosphatases

The dephosphorylation of phosphorylase kinase by four rabbit skeletal muscle protein phosphatases was studied. The four enzymes used were preparations of protein phosphatases C-I, C-II, H-I, and H-II. Phosphatases C-I, C-II, and H-II were obtained as homogeneous preparations using procedures previously developed. Phosphatase H-I was purified 644-fold from rabbit skeletal muscle for the purposes of this study, and was the major phosphorylase phosphatase activity in the tissue extract. Phosphatases C-I and H-I were relatively specific for removal of the beta subunit phosphate of phosphorylase kinase, this occurring at rates approximately 100 times more rapidly than the removal of the alpha subunit phosphate. In contrast, phosphatases C-II and H-II readily dephosphorylated both the alpha and beta subunits, although the alpha subunit phosphate release occurred at rates about twice that of the beta subunit phosphate. These studies show that skeletal muscle contains two phosphatases capable of acting on phosphorylase kinase, and that these have different specificities as represented by phosphatases H-I and C-I on the one hand, and phosphatases C-II and H-II on the other hand. These studies also provided unequivocal evidence that dephosphorylation of the beta subunit of phosphorylase kinase is solely involved in the inactivation of the cAMP-dependent protein kinase-activated enzyme. When autophosphorylated phosphorylase kinase was used as the substrate, the four phosphatases displayed similar general specificities as they did toward the cAMP-dependent protein kinase-activated enzyme. With none of the phosphatases examined was there any evidence that alpha subunit phosphorylation affected the rate of beta subunit dephosphorylation.     

The major type-1 protein phosphatase catalytic subunits are the same gene products in rabbit skeletal muscle and rabbit liver

The catalytic subunit of protein phosphatase-1 (PP1) isolated from rabbit liver had the same electrophoretic mobility as, and yielded peptide maps identical to those of the 33 kDa form of rabbit skeletal muscle PP1. The predicted amino-acid sequences of PP1 obtained from three rabbit liver cDNA clones were identical to that of PP1 alpha from rabbit skeletal muscle. These findings suggest that the distinctive substrate specificities and regulatory properties of hepatic and skeletal muscle type-1 protein phosphatases are not conferred by the catalytic subunits themselves, but by regulatory subunits that are complexed to the catalytic subunits in vivo.     

Ligand effects on the dephosphorylation of heart and skeletal muscle specific phosphorylases

Pseudo first order rate constants were determined for the dephosphorylation of heart and skeletal muscle specific phosphorylase a isoenzymes isolated from rabbit and pig using rabbit muscle phosphorylase phosphatase (mol. wt 34,000). The rate constants determined in the absence of ligands, were 4-5 fold lower for heart specific phosphorylases than for skeletal muscle specific ones. Glucose 6-phosphate (0.5-1 mM) enhances the rate of dephosphorylation of heart specific isophosphorylases 3-fold and suspends inhibition by 10(-5) M AMP, however, it has no significant effect on the dephosphorylation of skeletal muscle specific enzymes under the same conditions. Our data support characteristic functional differences between heart and skeletal muscle specific phosphorylases both in rabbit and pig.     

Function of the phosphate group of pyridoxal 5'-phosphate in the glycogen phosphorylase reaction

To understand the catalytic mechanism of glycogen phosphorylase (EC 2.4.1.1), pyridoxal(5')phospho(1)-beta-D-glucose was synthesized and examined as a hypothetical intermediate in the catalysis. Pyridoxal phosphoglucose bound stoichiometrically to the cofactor site of rabbit muscle phosphorylase b in a similar mode of binding to the natural cofactor, pyridoxal 5'-phosphate. The rate of binding of pyridoxal phosphoglucose was only 1/100 compared with that of pyridoxal phosphate. The enzyme reconstituted with pyridoxal phosphoglucose showed no enzymatic activity at all even after prolonged incubation of the enzyme with substrates and activator. The present data would contradict participation of the phosphate group of pyridoxal phosphate in a covalent glucosyl-enzyme intermediate even if the covalent intermediate was formed during the catalysis.     

Catalytic site of rabbit glycogen synthase isozymes. Identification of an active site lysine close to the amino terminus of the subunit

Rabbit skeletal muscle glycogen synthase was inhibited by pyridoxal 5'-phosphate and irreversibly inactivated after sodium borohydride reduction of the enzyme-pyridoxal-P complex. The irreversible inactivation by pyridoxal-P was opposed by the presence of the substrate UDP-glucose. With [3H]pyridoxal-P, covalent incorporation of 3H label into the enzyme could be monitored. UDP-glucose protected against 3H incorporation, whereas glucose-6-P was ineffective. Peptide mapping of tryptic digests indicated that two distinct peptides were specifically modified by pyridoxal-P. One of these peptides contained the NH2-terminal sequence of the glycogen synthase subunit. Chymotrypsin cleavage of this peptide resulted in a single-labeled fragment with the sequence: Glu-Val-Ala-Asn-(Pyridoxal-P-Lys)-Val-Gly-Gly-Ile-Tyr. This sequence is identical to that previously reported (Tagaya, M., Nakano, K., and Fukui, T. (1985) J. Biol. Chem. 260. 6670-6676) for a peptide specifically modified by a substrate analogue and inferred to form part of the active site of the enzyme. Sequence analysis revealed that the modified lysine was located at residue 38 from the NH2 terminus of the rabbit muscle glycogen synthase subunit. An analogous tryptic peptide obtained from the rabbit liver isozyme displayed a high degree of sequence homology in the vicinity of the modified lysine. We propose that the extreme NH2 terminus of the glycogen synthase subunit forms part of the catalytic site, in close proximity to one of the phosphorylated regions of the enzyme (site 2, serine 7). In addition, the work extends the known NH2-terminal amino acid sequences of both the liver and muscle glycogen synthase isozymes.     

Isolation and comparative studies of glycogen phosphorylase isoenzymes from lymphocytes and polymorphonuclear leucocytes

Two glycogen phosphorylase isoenzymes have been identified in pig lymphocytes and polymorphonuclear leucocytes by DEAE cellulose chromatography. Both isophosphorylases have been further purified by affinity chromatography on Sepharose-AMP to almost homogeneity. The purified isophosphorylases were composed of subunits of molecular weight similar to the muscle and liver monomers. Isophosphorylase I was more related to the liver enzyme than isophosphorylase II based on immuno-inhibition experiments. Both isoenzymes were markedly different from liver and muscle phosphorylases in their activation by AMP, sodium sulfate and 1,2-dimethoxyethane.     

Liver phosphorylase b kinase. Cyclic-AMP-mediated activation and properties of the partially purified rat-liver enzyme.

Phosphorylase b kinase was extensively purified from rat liver. It was located in a form which could be activated 20--30-fold by a preincubation with adenosine 3':5'-monophosphate (cyclic AMP) and ATP-Mg. This activation was time-dependent, and was paralleled by a simultaneous incorporation of 32P from [gamma-32P]ATP into two polypeptides which comigrated in sodium dodecyl sulfate gel electrophoresis with the alpha and beta subunits of rabbit skeletal muscle phosphorylase b kinase. The liver enzyme was eluted from Sepharose 4B and Bio-Gel A-50m columns at the same place as muscle phosphorylase b kinase, which is indicative of a molecular weight of 1.3 x 10(6). After activation, the most purified liver preparation had a specific activity about 10-fold less than the homogeneous muscle enzyme at pH 8.2. The inactive enzyme form had a pronounced pH optimum around pH 6.0, whereas the activated form was mostly active above neutral pH. The activation of the enzyme reduced the Km for its substrate phosphorylase b severalfold. Liver phosphorylase b kinase was shown to be partially dependent on Ca2+ ions for its activity: addition of 0.5 mM [ethylenebis-(oxoethylenenitrilo)]tetraacetic acid (EGTA) to the phosphorylase b kinase assay increased the Km for phosphorylase b about twofold for both the inactive and the activated form of liver phosphorylase b kinase, but affected the V of the inactive species only.     

Activation of human liver glycogen phosphorylase by alteration of the secondary structure and packing of the catalytic core

Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate, which enters glycolysis to fulfill the energetic requirements of the organism. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. To support inhibitor design, we determined the crystal structures of the active and inactive forms of human liver glycogen phosphorylase a. During activation, forty residues of the catalytic site undergo order/disorder transitions, changes in secondary structure, or packing to reorganize the catalytic site for substrate binding and catalysis. Knowing the inactive and active conformations of the liver enzyme and how each differs from its counterpart in muscle phosphorylase provides the basis for designing inhibitors that bind preferentially to the inactive conformation of the liver isozyme.     

The protein phosphatases involved in cellular regulation. Antibody to protein phosphatase-2A as a probe of phosphatase structure and function

Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B and 2C. The antibody was used to establish the following points. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complexed with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. The catalytic subunits of protein phosphatase-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylase, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis.     

Use of 6-fluoroderivatives of pyridoxal and pyridoxal phosphate in the study of the coenzyme function in glycogen phosphorylase

6-Fluoropyridoxal phosphate (6-FPLP) has been synthesized. Its properties were studied, and it was used, along with 6-fluoropyridoxal (6-FPAL), to reconstitute apophosphorylase b. Kinetic studies of the resulting enzymes showed that phosphorylases reconstituted with 6-FPLP and 6-FPAL have characteristics similar to those of native and pyridoxal enzymes, respectively, except that the former two enzymes have lower Vmax values. 19F NMR and UV spectra of 6-FPLP phosphorylase showed that the coenzyme forms a neutral enolimine Schiff base. Because the UV and fluorescence spectra of 6-FPLP phosphorylase are comparable to those obtained with native phosphorylase, it further confirms the postulate that pyridoxal phosphate forms a neutral enolimine Schiff base in phosphorylase. The results suggest that the 3-OH group is protonated and the pyridine nitrogen unprotonated in both 6-FPLP phosphorylase and native enzyme. 19F NMR study of 6-FPLP- and 6-FPAL-reconstituted phosphorylases in the inactive and active states indicates that the protein structure near the coenzyme binding site undergoes certain changes when these enzymes are activated by the substrates and AMP. The comparison of the properties of 6-FPLP-reconstituted and native phosphorylases implies that the ring nitrogen of the coenzyme PLP in phosphorylase may interact with the protein during catalysis, and this interaction is important for efficient catalysis by phosphorylase.     

Separation and identification of type 1 and type 2 protein phosphatases from rabbit reticulocyte lysates

Protein phosphatase type 1 and type 2 activities (designated PP-1 and PP-2, respectively) from rabbit reticulocyte lysates have been identified and characterized based on criteria previously established for similar activities in rabbit skeletal muscle and rabbit liver. These include (a) chromatographic separation on DEAE-cellulose, (b) substrate specificity toward glycogen phosphorylase a and the alpha- and beta-subunits of phosphorylase kinase, (c) differential sensitivity to the heat-stable protein phosphatase inhibitors-1 and -2, and (d) sensitivity to MgATP. When total lysate phosphatases are assayed in the presence of 1 mM MnCl2, protein phosphatase type 2 represents 84% of lysate phosphorylase phosphatase activity. However, when phosphatase assays are carried out with MgATP concentrations similar to those in the lysate, type 2 activity is diminished, and the levels of type 1 (41%) and type 2 (59%) phosphatase activities are comparable. A small proportion (6%) of total lysate phosphatase is tightly bound to the ribosomes, where type 1 phosphatase predominates. At least five species of protein phosphatases can be identified in lysates. These constitute two forms of protein phosphatase type 1, one of which (designated FC) is dependent on MgATP and a lysate activator protein FA; both FC and FA have been identified previously in skeletal muscle. Three species of protein phosphatase type 2 have been identified and designated PP-2B, PP-2A1, and PP-2A2 based on criteria recently established for rabbit skeletal muscle and rabbit liver phosphatases, which display similar phosphatase profiles. Lysate protein phosphatases types 1, FC, 2A1, and 2A2 can all act on phosphorylase a and the alpha- (type 2) or beta-(type 1) subunit of phosphorylase kinase. PP-2B, a Ca2+/calmodulin-dependent phosphatase, specifically dephosphorylates the alpha-subunit of phosphorylase kinase, but does not act on phosphorylase alpha. The heat-stable protein phosphatase inhibitor-2 from skeletal muscle completely blocks the activity of the two type 1 phosphatases (PP-1, FC), but has no effect on the three species of type 2 protein phosphatase. A preliminary assay of the two heat-stable phosphatase inhibitors in lysates indicates significant levels of inhibitor-2, but little or no detectable inhibitor-1.     

Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction.

Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that the active sites of glutamate aspartate transaminase function independently and a compulsory flip-flop mechanism is not involved.     

Temperature-sensitive production of rabbit muscle glycogen phosphorylase in Escherichia coli

In order to understand how allosteric switches regulate both the catalytic activity and molecular interactions of glycogen phosphorylase, it is necessary to design and analyze variant proteins that test hypotheses about the structural details of the allosteric mechanism. Essential to such an investigation is the ability to obtain large amounts of variant proteins. We developed a system for obtaining milligram amounts (greater than 20 mg/l) of rabbit muscle phosphorylase from bacteria. Phosphorylase aggregates as inactive protein when a strong bacterial promoter is used under full inducing conditions and normal growth conditions. However, when the growth temperature of bacteria expressing phosphorylase is reduced to 22 degrees C we obtain active muscle phosphorylase. The degree to which the induced expression of phosphorylase protein is temperature sensitive depends on the strain of bacteria used. New assay and purification methods were developed to allow rapid purification of engineered phosphorylase proteins from bacterial cultures. The rabbit muscle phosphorylase obtained from the bacterial expression system is enzymatically identical to the enzyme purified from rabbit muscle. The expressed protein crystallizes in the same conditions used for growing crystals of protein from rabbit muscle and the crystal form is isomorphous. Rabbit muscle phosphorylase is one of the largest oligomeric mammalian enzymes successfully expressed in Escherichia coli. Our results indicate that optimization of a combination of growth and induction conditions will be important in the expression of other heterologous proteins in bacteria.