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.     

Control of phosphorylase b conformation by a modified cofactor: crystallographic studies on R-state glycogen phosphorylase reconstituted with pyridoxal 5''-diphosphate.

Previous crystallographic studies on glycogen phosphorylase have described the different conformational states of the protein (T and R) that represent the allosteric transition and have shown how the properties of the 5'-phosphate group of the cofactor pyridoxal phosphate are influenced by these conformational states. The present work reports a study on glycogen phosphorylase b (GPb) complexed with a modified cofactor, pyridoxal 5'-diphosphate (PLPP), in place of the natural cofactor. Solution studies (Withers, S.G., Madsen, N.B., & Sykes, B.D., 1982, Biochemistry 21, 6716-6722) have shown that PLPP promotes R-state properties of the enzyme indicating that the cofactor can influence the conformational state of the protein. GPb complexed with pyridoxal 5'-diphosphate (PLPP) has been crystallized in the presence of IMP and ammonium sulfate in the monoclinic R-state crystal form and the structure refined from X-ray data to 2.8 A resolution to a crystallographic R value of 0.21. The global tertiary and quaternary structure in the vicinity of the Ser 14 and the IMP sites are nearly identical to those observed for the R-state GPb-AMP complex. At the catalytic site the second phosphate of PLPP is accommodated with essentially no change in structure from the R-state structure and is involved in interactions with the side chains of two lysine residues (Lys 568 and Lys 574) and the main chain nitrogen of Arg 569. Superposition of the T-state structure shows that were the PLPP to be incorporated into the T-state structure there would be a close contact with the 280s loop (residues 282-285) that would encourage the T to R allosteric transition. The second phosphate of the PLPP occupies a site that is distinct from other dianionic binding sites that have been observed for glucose-1-phosphate and sulfate (in the R state) and for heptulose-2-phosphate (in the T state). The results indicate mobility in the dianion recognition site, and the precise position is dependent on other linkages to the dianion. In the modified cofactor the second phosphate site is constrained by the covalent link to the first phosphate of PLPP. The observed position in the crystal suggests that it is too far from the substrate site to represent a site for catalysis.     

Segmental mobility in glycogen phosphorylase b

The dynamics and structuredness of the pyridoxal 5'-phosphate-binding region in glycogen phosphorylase b (EC 2.4.1.1) has been investigated with different techniques of fluorescence spectroscopy. Fluorescence polarization data of the thermal Perrin plot indicate some mobility in the cofactor binding site, while the isothermic measurements (at 20 degrees C, in high-viscosity solvents) demonstrate that the mobile unit carrying the emission oscillator is practically insensitive to the external viscosity. Characteristics of the thermal Perrin plots obtained for both native and reduced phosphorylase b can be interpreted either as a freely moving cofactor in a medium of high viscosity (0.3 P) or as the motion of a unit larger than a lysine-bonded pyridoxal 5'-phosphate in a medium with the viscosity of water. Data for acrylamide quenching and time-resolved fluorescence measurements suggest that the latter interpretation should valid. These data also suggest a tightly packed microenvironment around the pyridoxal moiety.     

Activator anion binding site in pyridoxal phosphorylase b: the binding of phosphite, phosphate, and fluorophosphate in the crystal.

It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5'-phosphate (Change YC. McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 A resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5'-phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 A). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis.     

Refined crystal structure of the phosphorylase-heptulose 2-phosphate-oligosaccharide-AMP complex

The crystal structure of phosphorylase b-heptulose 2-phosphate complex with oligosaccharide and AMP bound has been refined by molecular dynamics and crystallographic least-squares with the program XPLOR. Shifts in atomic positions of up to 4 A from the native enzyme structure were correctly determined by the program without manual intervention. The final crystallographic R value for data between 8 and 2.86 A resolution is 0.201, and the overall root-mean-square difference between the native and complexed structure is 0.58 A for all protein atoms. The results confirm the previous observation that there is a direct hydrogen bond between the phosphate of heptulose 2-phosphate and the pyridoxal phosphate 5'-phosphate group. The close proximity of the two phosphates is stabilized by an arginine residue, Arg569, which shifts from a site buried in the protein to a position where it can make contact with the product phosphate. There is a mutual interchange in position between the arginine and an acidic group, Asp283. These movements represent the first stage of the allosteric response which converts the catalytic site from a low to a high-affinity binding site. Communication of these changes to other sites is prevented in the crystal by the lattice forces, which also form the subunit interface. The constellation of groups in the phosphorylase transition state analogue complex provides a structural basis for understanding the catalytic mechanism in which the cofactor pyridoxal phosphate 5'-phosphate group functions as a general acid to promote attack by the substrate phosphate on the glycosidic bond when the reaction proceeds in the direction of glycogen degradation. In the direction of glycogen synthesis, stereoelectronic effects contribute to the cleavage of the C-1-O-1 bond. In both reactions the substrate phosphate plays a key role in transition state stabilization. The details of the oligosaccharide, maltoheptaose, interactions with the enzyme at the glycogen storage site are also described.     

Pyridoxal phosphate site in glycogen phosphorylase b: structure in native enzyme and in three derivatives with modified cofactors

The detailed environment of the essential cofactor pyridoxal 5'-phosphate in glycogen phosphorylase b, resulting from crystallographic refinement at 1.9-A resolution, is described. The pyridoxal ring is buried in a nonpolar site containing three aromatic rings while the 5'-phosphate group is highly solvated and makes only three direct contacts to the protein. The pyridine nitrogen interacts via a water with protein atoms [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. The crystal structures of three active derivatives of phosphorylase reconstituted with 5'-deoxypyridoxal 5'-methylenephosphonate (PDMP), 6-fluoropyridoxal 5'-phosphate (6-FPLP), and pyridoxal (PL) in place of the natural cofactor have been determined at 2.5-A resolution. The results for PDMP-phosphorylase show a closer proximity of the phosphonate group to the NZ atom of a lysine (Lys-574) than that observed in the native enzyme, consistent with 31P NMR studies that have shown a change in ionization state of the phosphonate group compared to the native cofactor phosphate. The replacement of the polar 5'-ester linkage by a CH2 group results in a small shift of a water and its hydrogen-bonded tyrosine (Tyr-648). In 6-FPLP-phosphorylase the fluorine is accommodated with no significant change in structure. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Observations on the pyridoxal 5'-phosphate inhibition of DNA polymerases.

Pyridoxal 5'-phosphate at concentrations greater than 0.5 mM inhibits polymerization of deoxynucleoside triphosphate catalyzed by a variety of DNA polymerases. The requirement for a phosphate as well as aldehyde moiety of pyridoxal phosphate for inhibition to occur is clearly shown by the fact that neither pyridoxal nor pyridoxamine phosphate are effective inhibitors. Since the addition of nonenzyme protein or increasing the amount of template primer exerted no protective effect, there appears to be specific affinity between pyridoxal phosphate and polymerase protein. The deoxynucleoside triphosphates, however, could reverse the inhibition. The binding of pyridoxal 5'-phosphate to enzyme appears to be mediated through classical Schiff base formation between the pyridoxal phosphate and the free amino group(s) present at the active site of the polymerase protein. Kinetic studies indicate that inhibition by pyridoxal phosphate is competitive with respect to substrate deoxynucleoside triphosphate(s).     

Cysteine synthase of an extremely thermophilic bacterium,Thermus thermophilus HB8

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source. Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography). The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism. Molecular masses of 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure. The enzyme was stable at 70 degrees C at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80 degrees C with 10 mm dithiothreitol. The enzyme was also quite stable at pH 8-12 (50 degrees C, 30 min). It had an apparent Km of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a Vmax of 435 micromol/min/mg of protein. The apparent Km for sulfide was approximately 50 microM (with 20 mM acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine. The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5'-phosphate as a cofactor. The apo-enzyme showed an apparent Km of 29 microM for the cofactor at pH 8. Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme. The meaning of a very high enzyme content in the cell is discussed.     

Photoaffinity labeling of the glucagon receptor with a new glucagon analog

The flash excitation of the pyridoxal 5'-phosphate cofactor of glycogen phosphorylase b by an ultraviolet laser produces a transient state from a proton transfer of the bound cofactor. The rate of decay of this transient state is sensitive to the ionization state of the cofactor. This proved a useful probe for the ionization state of the 5'-phosphate group of the cofactor on the binding by the enzyme of various substrates. The decay rate data show, for the binding of glucose 1-phosphate, a partially negative 5'-HPO4- and evidence for a PO4-PO4 interaction. The data is interpreted in terms of a dynamic shift of substrates at the active site.     

Relationships between structure, function and stability for pyridoxal 5'-phosphate-dependent starch phosphorylase from Corynebacterium callunae as revealed by reversible cofactor dissociation studies.

Using 0.4 m imidazole citrate buffer (pH 7.5) containing 0.1 mm l-cysteine, homodimeric starch phosphorylase from Corynebacterium calluane (CcStP) was dissociated into native-like folded subunits concomitant with release of pyridoxal 5'-phosphate and loss of activity. The inactivation rate of CcStP under resolution conditions at 30 degrees C was, respectively, four- and threefold reduced in two mutants, Arg234-->Ala and Arg242-->Ala, previously shown to cause thermostabilization of CcStP [Griessler, R., Schwarz, A., Mucha, J. & Nidetzky, B. (2003) Eur. J. Biochem.270, 2126-2136]. The proportion of original enzyme activity restored upon the reconstitution of wild-type and mutant apo-phosphorylases with pyridoxal 5'-phosphate was increased up to 4.5-fold by added phosphate. The effect on recovery of activity displayed a saturatable dependence on the phosphate concentration and results from interactions with the oxyanion that are specific to the quarternary state. Arg234-->Ala and Arg242-->Ala mutants showed, respectively, eight- and > 20-fold decreased apparent affinities for phosphate (K(app)), compared to the wild-type (K(app) approximately 6 mm). When reconstituted next to each other in solution, apo-protomers of CcStP and Escherichia coli maltodextrin phosphorylase did not detectably associate to hybrid dimers, indicating that structural complementarity among the different subunits was lacking. Pyridoxal-reconstituted CcStP was inactive but approximately 60% and 5% of wild-type activity could be rescued at pH 7.5 by phosphate (3 mm) and phosphite (5 mm), respectively. pH effects on catalytic rates were different for the native enzyme and pyridoxal-phosphorylase bound to phosphate and could reflect the differences in pK(a) values for the cofactor 5'-phosphate and the exogenous oxyanion.     

Mitochondrial aspartate aminotransferase-independent function of the catalytic binding sites.

The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5-phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal-5-P per dimer, three forms of the enzyme may exist in solution: apoenzyme-2 pyridoxal 5'-phosphate, apoenzyme-1 pyridoxal 5'-phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme-2 pyridoxal 5'-phosphate/apoenzyme-1 pytidoxal 5'-phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal-5-P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme-1 pyridoxal 5'-phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4-pyridoxic-5-P (KD equals 1.1 times 10- minus 6 M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by two independent methods, lend strong support to the hypothesis that the two subunits of the enzyme function independently.     

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with pyridoxal 5'-phosphate at 2.0 A resolution.

Escherichia coli pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate by the FMN oxidation of pyridoxine 5'-phosphate forming FMNH(2) and H(2)O(2). Recent studies have shown that in addition to the active site, pyridoxine 5'-phosphate oxidase contains a non-catalytic site that binds pyridoxal 5'-phosphate tightly. The crystal structure of pyridoxine 5'-phosphate oxidase from E. coli with one or two molecules of pyridoxal 5'-phosphate bound to each monomer has been determined to 2.0 A resolution. One of the pyridoxal 5'-phosphate molecules is clearly bound at the active site with the aldehyde at C4' of pyridoxal 5'-phosphate near N5 of the bound FMN. A protein conformational change has occurred that partially closes the active site. The orientation of the bound pyridoxal 5'-phosphate suggests that the enzyme catalyzes a hydride ion transfer between C4' of pyridoxal 5'-phosphate and N5 of FMN. When the crystals are soaked with excess pyridoxal 5'-phosphate an additional molecule of this cofactor is also bound about 11 A from the active site. A possible tunnel exists between the two sites so that pyridoxal 5'-phosphate formed at the active site may transfer to the non-catalytic site without passing though the solvent.     

Studies on the changes in protein fluorescence and enzymic activity of aspartate aminotransferase on binding of pyridoxal 5′-phosphate

1. The alpha and beta subforms of aspartate aminotransferase were purified from pig heart. 2. The alpha subform contained 2mol of pyridoxal 5'-phosphate. The apo-(alpha subform) could be fully reactived by combination with 2mol of cofactor. 3. The protein fluorescence of the apo-(alpha subform) decreased non-linearly with increase in enzyme activity and concentration of bound cofactor. 4. It is concluded that the enzyme activity/mol of bound cofactor is largely independent of the number of cofactors bound to the dimer. 5. The beta subform had approximately half the specific enzyme activity of the alpha subform, and contained an average of one active pyridoxal 5'-phosphate molecule per molecule, which could be removed by glutamate, and another inactive cofactor which could only be removed with NaOH. 6. On recombination with pyridoxal 5'-phosphate the protein fluorescence of the apo-(beta subform) decreased linearly, showing that each dimeric enzyme molecule contained one active and one inactive bound cofactor. 7. The results are not consistent with a flip-flop mechanism for this enzyme.     

Functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate in phosphorylase: a study using pyridoxal-reconstituted enzyme as a model system

Pyridoxal-reconstituted phosphorylase was used as a model system to study the possible functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate (PLP) in rabbit muscle glycogen phosphorylase. Kinetic study was conducted by using competitive inhibitors of phosphite, an activator, and alpha-D-glucopyranose 1-phosphate (glucose-1-P) to study the relationship between the PLP phosphate and the binding of glucose-1-P to phosphorylase. Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy of fluorophosphate bound to pyridoxal phosphorylase showed that its ionization state did not change during enzymatic catalysis. Evaluation of the apparent kinetic parameters for the activation of pyridoxal phosphorylase with different analogues having varied pKa2 values demonstrated a dependency of KM on pKa2. Molybdate, capable of binding as chelates in a trigonal-bipyramidal configuration, was tested for its inhibitory property with pyridoxal phosphorylase. On the basis of the results in this study, several conclusions may be drawn: (1) The bound phosphite in pyridoxal phosphorylase and, possibly, the 5'-phosphoryl group of PLP in native phosphorylase do not effect the glucose-1-P binding. (2) One likely function of the 5'-phosphoryl group of PLP in native phosphorylase is acting as an anchoring point to hold the PLP molecule and/or various amino acid side chains in a proper orientation for effective catalysis. (3) The force between the PLP phosphate and its binding site in phosphorylase is mainly electrostatic; a change of ionization state during catalysis is unlikely. (4) Properties of the central atoms of different anions are important for their effects as either activators or inhibitors of pyridoxal phosphorylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of glutamate decarboxylase from Solanum tuberosum

Glutamate decarboxylase has been purified from potato tubers. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Gel filtration on Sephadex G-200 gave a relative molecular mass Mr, of 91 000 for the native enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a subunit Mr of 43 000. Thus the enzyme appears to be a dimer of identical subunits. It has 2 mol pyridoxal 5'-phosphate/mol protein, which could not be removed by exhaustive dialysis or gel filtration on Sephadex G-25. The enzyme has an absorption maximum at 370 nm in sodium phosphate buffer, pH 5.8. Reduction of the enzyme with sodium borohydride abolished the absorption maximum at 370 nm with attendant loss of catalytic activity. The enzyme exhibited pH-dependent spectral changes. The enzyme was specific for L-glutamate and could not decarboxylate other amino acids tested. The enzyme was maximally active at pH 5.8 and a temperature of 37 degrees C. Isoelectric focussing gave a pI of 4.7 Km values for L-glutamate and pyridoxal 5'-phosphate were 5.6 mM and 2 microM respectively. Thiol-directed reagents and heavy metal ions inhibited the enzyme, indicating that an -SH group is required for activity. The nature of the functional groups at the active site of the enzyme was inferred from competitive inhibition studies. L-Glutamate promoted inactivation of the enzyme caused by decarboxylation-dependent transamination was demonstrated. The characteristics of potato enzyme were compared with enzyme from other sources.     

Conformational changes and stabilization induced by phosphate binding to 5′-methylthioadenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus

The effect of phosphate, its analogues, and other substrates on structural features of recombinant 5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was investigated. Phosphate was found to exert a significant stabilizing effect on the protein against the inactivation caused by temperature, sodium dodecyl sulfate (SDS), urea, and proteolytic enzymes. In the presence of 100 mM phosphate: (i) the apparent transition temperature (Tm) of recombinant SsMTAP increased from 111 degrees to 118 degrees C; and (ii) the enzyme still retained 40% and 30% activity, respectively, after 30 min of incubation at 90 degrees C with 2% SDS or 8 M urea. The structure modification of SsMTAP by phosphate binding was probed by limited proteolysis with subtilisin and proteinase K and analysis of polypeptide fragments by SDS-PAGE. The binding of the phosphate substrate protected SsMTAP against protease inactivation, as proven by the disappearance of a previously accessible proteolytic cleavage site that was localized in the N-terminal region of the enzyme. The conformational changes of SsMTAP induced by phosphate and ribose-1-phosphate were analyzed by fluorescence spectroscopy, and modifications of the protein intrinsic fluorophore exposure, as a consequence of substrate binding, were evidenced.     

Oxyanion-Mediated Protein Stabilization: Differential Roles of Phosphate for Preventing Inactivation of Bacterial α-Glucan Phosphorylases

Maltodextrin phosphorylase (MalP) from Escherichia coli and starch phosphorylase (StP) from Corynebacterium callunae are significantly stabilized in the presence of phosphate against inactivation by elevated temperature or urea. The stabilizing effect of phosphate was observed at ion concentrations below 50 mM. Therefore, it is probably due to preferential binding of phosphate to the folded conformations of the phosphorylases. For StP, phosphate binding inhibited the dissociation of the active-site cofactor pyridoxal 5′-phosphate. Phosphate-liganded StP was at least 500-fold more stable at 60d`C than the free enzyme at the same temperature. It showed an apparent transition midpoint of 5.2 M for irreversible denaturation by urea, and this midpoint was increased by a denaturant concentration of 4M relative to the corresponding transition midpoint of free StP in urea. The mechanisms of inactivation and denaturation of MalP at 45d`C and by urea involve formation of a cofactor-containing, insoluble protein aggregate. Under denaturing conditions, phosphate was shown to inhibit aggregation of the reversibly inactivated MalP dimer.     

Substrate-cofactor interactions for glycogen phosphorylase b: a binding study in the crystal with heptenitol and heptulose 2-phosphate

The structural relationships between substrate and pyridoxal phosphate in glycogen phosphorylase b (EC 2.4.1.1) have been studied by X-ray diffraction experiments at 3-A resolution. Recent work [Klein, H. W., Im, M. J., & Helmreich, E. J. M. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, A. E., Ed.) pp 147-160, Liss, New York] has shown that phosphorylase in the presence of inorganic phosphate catalyzes the conversion of heptenitol to heptulose 2-phosphate. The latter compound is a dead-end product and a most potent inhibitor (Ki = 14 microM). The X-ray diffraction studies show that heptenitol binds at the catalytic site of phosphorylase in a position essentially identical with that observed for the glucopyranose moiety of glucose 1-phosphate. Incubation of a phosphorylase b crystal for 50 h in a solution containing the substrates heptenitol and inorganic phosphate and the activators AMP and maltohetaose resulted in the formation of a phosphorylated product bound at the active site. The structure of this product, as analyzed by a difference Fourier synthesis at 3 A, is consistent with that of heptulose 2-phosphate. Analysis of the surrounding soak solution by thin-layer chromatography showed that heptulose 2-phosphate was produced under these conditions. Heptulose 2-phosphate binds with its glucopyranose moiety in the same position as that for glucose 1-phosphate, but there is a marked difference in phosphate positions. The presence of the methyl group in the beta-configuration in heptulose 2-phosphate forces a change in the torsion angle O5-C1-O1-P from 117 degrees as observe in glucose 1-phosphate to -136 degrees in heptulose 2-phosphate. The "down" position of the phosphate (with respect to the crystallographic z axis) results in a change in the distance between the 5'-phosphorus atom of the pyridoxal phosphate and the phosphorus atom of the substrate from 6.8 (with glucose 1-phosphate) to 4.5 A (with heptulose 2-phosphate). The closest distance between the phosphate oxygen of the cofactor and a phosphate oxygen of heptulose 2-phosphate is 2.7 A, and it is assumed that there must be a hydrogen bond between them. These observations are consistent with the NMR experiments reported in the preceding paper in which sharing of a proton between heptulose 2-phosphate and pyridoxal 5'-phosphate is observed [Klein, H.W., Im, M. J., Palm, D., & Helmreich, E. J. M. (1984) Biochemistry (preceding paper in this issue)].(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural changes accompanying the removal of pyridoxal 5'-phosphate from phosphorylase b.

The changes in physical properties accompanying the removal of pyridoxal 5'-phosphate from glycogen phosphorylase b have been examined. The apoenzyme retains a high degree of structural rigidity, as determined from the time decay of anisotropy. The bulk of the secondary structure remains intact, although a significant change in circular dichroism indicates some degree of alteration. The mobility of a sulfhydryl-linked spin label increases. The restoration of pyridoxal 5'-phosphate reverses this effect, with indication of interaction between subunits. One or more new binding sites for 1-anilinonaphthalene-8-sulfonate appear for the apoenzyme. The kinetics of the recombination of pyridoxal 5'-phosphate with the apoenzyme, as monitored by difference spectra, indicate a high activation energy for the process. The apoenzyme is a reversibly associating system at 20-30 degrees C, pH 7.0.     

Crystal structure at 2.4 A resolution of E. coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate

Serine hydroxymethyltransferase (EC 2.1.2.1), a member of the alpha-class of pyridoxal phosphate enzymes, catalyzes the reversible interconversion of serine and glycine, changing the chemical bonding at the C(alpha)-C(beta) bond of the serine side-chain mediated by the pyridoxal phosphate cofactor. Scission of the C(alpha)-C(beta) bond of serine substrate produces a glycine product and most likely formaldehyde, which reacts without dissociation with tetrahydropteroylglutamate cofactor. Crystal structures of the human and rabbit cytosolic serine hydroxymethyltransferases (SHMT) confirmed their close similarity in tertiary and dimeric subunit structure to each other and to aspartate aminotransferase, the archetypal alpha-class pyridoxal 5'-phosphate enzyme. We describe here the structure at 2.4 A resolution of Escherichia coli serine hydroxymethyltransferase in ternary complex with glycine and 5-formyl tetrahydropteroylglutamate, refined to an R-factor value of 17.4 % and R(free) value of 19.6 %. This structure reveals the interactions of both cofactors and glycine substrate with the enzyme. Comparison with the E. coli aspartate aminotransferase structure shows the distinctions in sequence and structure which define the folate cofactor binding site in serine hydroxymethyltransferase and the differences in orientation of the amino terminal arm, the evolution of which was necessary for elaboration of the folate binding site. Comparison with the unliganded rabbit cytosolic serine hydroxymethyltransferase structure identifies changes in the conformation of the enzyme, similar to those observed in aspartate aminotransferase, that probably accompany the binding of substrate. The tetrameric quaternary structure of liganded E. coli serine hydroxymethyltransferase also differs in symmetry and relative disposition of the functional tight dimers from that of the unliganded eukaryotic enzymes. SHMT tetramers have surface charge distributions which suggest distinctions in folate binding between eukaryotic and E. coli enzymes. The structure of the E. coli ternary complex provides the basis for a thorough investigation of its mechanism through characterization and structure determination of site mutants.