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PubMed1992

Cooperative binding is not required for activation of muscle phosphorylase.

Muscle and liver glycogen phosphorylase isozymes differ in their responsiveness to the activating ligand AMP. The muscle enzyme, which supplies glucose in response to strenuous activity, binds AMP cooperatively, and its enzymatic activity becomes greatly enhanced. The liver isozyme regulates the level of blood glucose, and AMP is not the primary activator. In muscle glycogen phosphorylase, the residue proline 48 links two secondary structural elements that bind AMP. This amino acid residue is replaced with a threonine in the liver isozyme; unlike the muscle enzyme, liver binds AMP noncooperatively, and the enzymatic activity is not greatly increased. We have substituted proline 48 in the muscle enzyme with threonine, alanine, and glycine and characterized the recombinant enzymes kinetically and structurally to determine if proline at this position is critical for cooperative AMP binding and activation. Importantly, all of the engineered enzymes were fully activated by phosphorylation, indicating that enzymatic activity was not compromised. Only the mutant enzyme with alanine at position 48 responds like the wild-type enzyme to the presence of AMP, indicating that proline is not absolutely required for full cooperative activation. The substitution of either threonine or glycine at this position, however, creates enzymes that no longer bind AMP cooperatively. The enzyme with threonine at position 48 further mimics the liver enzyme, in that the maximal enzymatic activity is also reduced. Significantly, the glycine substitution caused the enzyme to be fully activated by AMP, although binding was not cooperative. The hyperactivation of the glycine mutant by AMP suggests that the total free energy of activation has decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1993

Purine nucleoside phosphorylase. Catalytic mechanism and transition-state analysis of the arsenolysis reaction.

Purine nucleoside phosphorylase from calf spleen catalyzes the arsenolysis of inosine to form hypoxanthine and ribose 1-arsenate, which spontaneously hydrolyzes to ribose and arsenate. In the presence of H2(18)O, no 18O is incorporated into ribose, demonstrating that ribose 1-arsenate hydrolysis occurs by attack of water on the arsenic atom. Rapid reaction kinetics at 20 degrees C result in a biphasic rate curve with the first turnover occurring at a rate of 20 s-1 followed by a steady-state rate of 2 s-1. The product burst is consistent with rapid steps for substrate binding and arsenolysis followed by rate-limiting hypoxanthine release at a rate of 2 s-1. Purine nucleoside phosphorylase with bound [14C]inosine was mixed with excess unlabeled inosine and arsenate to determine relative rates for reaction or dissociation of bound inosine. The commitment factor (product formed/inosine released) was 0.19 at saturating arsenate, indicating that inosine binds to free enzyme and that bound inosine is not in thermodynamic equilibrium with free substrate. At neutral pH, kinetic isotope effects for the phosphorolysis reaction are small, indicating kinetic suppression. Kinetic isotope effects for arsenolysis were measured with [1'-3H]-, [2'-3H]-, [1'-14C]-, [9-15N]-, [4'-3H]-, and [5'-3H]inosine to provide experimental values of 1.118 +/- 0.003, 1.128 +/- 0.003, 1.022 +/- 0.005, 1.009 +/- 0.004, 1.007 +/- 0.003 and 1.028 +/- 0.004 respectively. Following correction for commitment factors, the intrinsic isotope effects were matched to a geometric transition-state model selected by bond-energy bond order vibrational analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Purification and physicochemical properties of starch phosphorylase from young banana leaves.

Starch phosphorylase from young banana leaves has been purified to homogeneity, as tested by disc polyacrylamide gel electrophoresis at various pHs and gel concentrations, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunodiffusion, and immunoelectrophoresis, using the techniques of ammonium sulfate fractionation, DEAE-cellulose chromatography, and filtration through Sephadex G-100 and Sephadex G-200. The molecular weight of the enzyme is found to be 45000 as determined by gel filtration chromatography over Sephadex G-200. The enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having the molecular weight 55000. The enzyme contains eight SH groups per mol of the enzyme. Unlike other 1,4-alpha-glucan phosphorylases, no evidence is found for the presence of pyridoxal 5'-phosphate as a prosthetic group of the enzyme. Of the various amino acids tested, only aromatic amino acids inhibited the enzyme activity. ADP, AMP, and 3',5'-AMP did not produce any effect on the enzyme activity whereas ATP and UDP-glucose proved to be inhibitors. The enzyme utilized starch, amylose, and glycogen as primers with equal efficiency whereas dextrin, amylopectin, maltotriose, and maltose were less effective as primers. Schardinger dextrin, cellulose, or sucrose could not be utilized as a primer. The enzyme showed absolute specificity for glucose 1-phosphate as a substrate, and this could not be replaced by glucose-6-phosphate, fructose-6-phosphate, fructose 1,6-bisphosphate, or ribose 5-phosphate.

Adenosine Triphosphate↗
PubMed1985

Kinetic studies of thymidine phosphorylase from mouse liver.

Initial velocity and product inhibition studies of thymidine phosphorylase from mouse liver revealed that the basic reaction mechanism of this enzyme is a rapid equilibrium random bi-bi mechanism with an enzyme-phosphate-thymine dead-end complex. Thymine displayed both substrate inhibition and nonlinear product inhibition, i.e., slope and intercept replots vs. 1/[thymine] were nonlinear, indicating that there is more than one binding site on the enzyme for thymine and that when thymine is bound to one of these sites, the enzyme is inhibited. Furthermore, both thymidine and phosphate showed "cooperative effects" in the presence of thymine at concentrations above 60 microM, suggesting that the enzyme may have multiple interacting allosteric and/or catalytic sites. The deoxyribosyl transferase reaction catalyzed by this enzyme is phosphate-dependent, requires nonstoichiometric amounts of phosphate, and can proceed by an "enzyme-bound" 2-deoxyribose 1-phosphate intermediate. These findings are in accord with the rapid equilibrium random bi-bi mechanism and demonstrate that deoxyribosyl transfer by this enzyme involves an indirect-transfer mechanism. These results strongly suggest that phosphorolysis and deoxyribosyl transfer are catalyzed by the same site on thymidine phosphorylase.

Animals↗
PubMed1986

Hydrogen bonding and specificity. Fluorodeoxy sugars as probes of hydrogen bonding in the glycogen phosphorylase-glucose complex.

The affinities of a large number of deoxy and fluorodeoxy sugars for the glucose binding site in glycogen phosphorylase have been measured, and polarities and relative strengths of the hydrogen bonds at each position have been predicted on the basis of these data. Comparison with the recently refined X-ray crystal structure of the phosphorylase-glucose complex shows a generally good correlation between predicted and observed bond strengths, vindicating this approach to the evaluation of hydrogen bonding. Estimates of the net contributions of hydrogen bonds of different types (neutral-neutral and neutral-charged) are essentially identical with those obtained by a complementary approach on the tyrosyl tRNA synthetase-substrate complex [Fersht, A. R., Shi, J. P., Knill-Jones, J., Lowe, D. M., Wilkinson, A. J., Blow, D. M., Brick, P., Cortes, P., Waye, M. M. Y., & Winter, G. (1985) Nature (London) 314, 235-238]. The carbohydrate binding site structure determined is compared with that recently determined for the arabinose binding protein.

Animals↗
PubMed1987

Function of pyridoxal 5'-phosphate in glycogen phosphorylase: a model study using 6-fluoro-5'-deoxypyridoxal- and 5'-deoxypyridoxal-reconstituted enzymes.

A new vitamin B6 analogue, 6-fluoro-5'-deoxypyridoxal (6-FDPL), was synthesized and characterized. This analogue, as well as 6-fluoropyridoxal (6-FPAL), 6-fluoropyridoxal phosphate (6-FPLP), and 6-fluoropyridoxine, showed positive heteronuclear 1H-19F nuclear Overhauser effects between the 5'-protons and the 6-fluorine. Apophosphorylase reconstituted with 6-FDPL showed 1% of the activity of the native enzyme in the presence of phosphite. The kinetic pattern, apparent pH optimum of activity, and the activity-temperature dependency of the 6-FDPL-enzyme were virtually identical with those of phosphorylase reconstituted with the parent compound, 6-FPAL [Chang, Y. C., & Graves, D. J. (1985) J. Biol. Chem. 260, 2709-2714], except the Km of phosphite toward the 6-FDPL-enzyme was 9 times higher than that with the 6-FPAL-enzyme and the 6-FDPL-enzyme showed a lower Vmax value. Phosphorylase reconstituted with 5'-deoxypyridoxal (DPL) also showed activity in the presence of phosphite. The kinetics and the temperature-activity dependency of this reconstituted enzyme were investigated. 19F nuclear magnetic resonance studies showed that the binding of glucose 1-phosphate to a 6-FDPL-enzyme-adenosine 5'-phosphate (AMP) complex shifted the 19F signal 0.6 ppm upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed a complex with glucose 1-phosphate [Chang, Y. C., Scott, R. D., & Graves, D. J. (1986) Biochemistry 25, 1932-1939].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Fluorinated and deoxygenated substrates as probes of transition-state structure in glycogen phosphorylase.

A series of deoxyfluoro- and deoxy-alpha-D-glucopyranosyl phosphates have been tested as substrates of rabbit muscle glycogen phosphorylase b. All are found to be utilized by the enzyme, but at substantially reduced rates. Values of Vm/Km for these analogues range from 10(2) to 10(5) times lower than that for the parent substrate. The large rate reductions are suggested to arise from a combination of intrinsic electronic effects and poorer binding of these substrates at the transition state. The data provide substantial evidence for an oxocarbonium-ion-like transition state. They also provide estimates of the strengths of hydrogen bonds to individual sugar hydroxyls at the transition state of the reaction. Further, comparison of such data with those obtained for glucose analogues binding as inhibitors to T-state phosphorylase suggests that these two glucose subsites are essentially identical; thus, the glucose pocket remains intact during the conformational transition associated with activation of the enzyme.

Animals↗
PubMed1990

Measurement of active-site homology between potato and rabbit muscle alpha-glucan phosphorylases through use of a linear free energy relationship.

The Michaelis-Menten parameters (Vmax and Km) for turnover of an extensive series of deoxy and deoxyfluoro derivatives of alpha-D-glucopyranosyl phosphate by the alpha-glucan phosphorylase from potato tuber have been determined. Very large rate reductions are observed as a consequence of each substitution, primarily due to losses in specific binding interactions, most likely hydrogen bonding, at the enzymic transition state. Comparison of the Vmax/Km values so determined with those measured for rabbit muscle alpha-glucan phosphorylase [Street et al. (1989) Biochemistry 28, 1581] reveals an astonishingly similar specificity, especially in light of the phylogenetic separation of their host organisms. This indicates that very similar hydrogen-bonding interactions between the enzyme and the substrate must be present at the transition states for the two enzymic reactions; therefore, they have very similar active sites. Quantitation of this similarity is achieved by plotting the logarithm of the Vmax/Km value for each substrate analogue with the potato enzyme against the same parameter for the muscle enzyme, yielding straight lines (p = 0.998 and 0.999) of slope 1.0 and 1.2 for the deoxy and deoxyfluoro substrates, respectively. Since the correlation coefficient of such plots is a direct measure of the similarity of the two transition-state complexes, thus of the enzyme active sites, it can be used as a measure of active-site homology between the two enzymes. The extremely high homology observed in this case is consistent with the observed sequence homology at the active site.

Animals↗
PubMed1990

Small-angle scattering studies show distinct conformations of calmodulin in its complexes with two peptides based on the regulatory domain of the catalytic subunit of phosphorylase kinase.

Small-angle X-ray and neutron scattering have been used to study the solution structures of calmodulin complexed with synthetic peptides corresponding to residues 342-366 and 301-326, designated PhK5 and PhK13, respectively, in the regulatory domain of the catalytic subunit of skeletal muscle phosphorylase kinase. The scattering data show that binding of PhK5 to calmodulin induces a dramatic contraction of calmodulin, similar to that previously observed when calmodulin is complexed with the calmodulin-binding domain peptide from rabbit skeletal muscle myosin light chain kinase. In contrast, calmodulin remains extended upon binding PhK13. In the presence of both peptides, calmodulin also remains extended. Apparently, the presence of PhK13 inhibits calmodulin from undergoing the PhK5-induced contraction. These data indicate that there is a fundamentally different type of calmodulin-target enzyme interaction in the case of the catalytic subunit of phosphorylase kinase compared with that for myosin light chain kinase.

Amino Acid Sequence↗
PubMed1990

Comparison of the binding of glucose and glucose 1-phosphate derivatives to T-state glycogen phosphorylase b.

The binding of T-state- and R-state-stabilizing ligands to the catalytic C site of T-state glycogen phosphorylase b has been investigated by crystallographic methods to study the interactions made and the conformational changes that occur at the C site. The compounds studied were alpha-D-glucose, 1, a T-state-stabilizing inhibitor of the enzyme, and the R-state-stabilizing phosphorylated ligands alpha-D-glucose 1-phosphate (2), 2-deoxy-2-fluoro-alpha-D-glucose 1-phosphate (3), and alpha-D-glucose 1-methylenephosphonate (4). The complexes have been refined, giving crystallographic R factors of less than 19%, for data between 8 and 2.3 A. Analysis of the refined structures shows that the glucosyl portions of the phosphorylated ligands bind in the same orientation as glucose and retain most of the interactions formed between glucose and the enzyme. However, the phosphates of the phosphorylated ligands adopt different conformations in each case; the stability of these conformations have been studied by using computational methods to rationalize the different binding modes. Binding of the phosphorylated ligands is accompanied by movement of C-site residues, most notably a shift of a loop out of the C site and toward the exterior of the protein. The C-site alterations do not include movement of Arg569, which has been observed in both the refined complex with 1-deoxy-D-gluco-heptulose 2-phosphate (5) [Johnson, L. N., et al (1990) J. Mol. Biol. 211, 645-661] and in the R-state enzyme [Barford, D. & Johnson, L. N. (1989) Nature 340, 609-616]. Refinement of the ligand complexes has also led to the observation of additional electron density for residues 10-19 at the N-terminus which had not previously been localized in the native structure. The conformation of this stretch of residues is different from that observed in glycogen phosphorylase a.

Animals↗
PubMed1978

Sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase including the sites of covalent and allosteric control.

The sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase (EC 2.4.1.1) has been determined. Limited proteolysis of native phosphorylase b (841 residues, subunit molecular weight 97 412) by subtilisin BPN', Streptomyces alkaline protease, or elastase yielded two large segments (light and heavy). The light segment isolated from the subtilisin digest was cleaved at methionyl bonds with cyanogen bromide to yield eight major fragments and two minor overlapping fragments. The alignment of the major fragments was obtained by analysis of the two minor fragments, of five tryptic peptides containing methionine and of one large fragment generated by cleavage of an aspartylproline bond. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule identified the sites susceptible to limited proteolysis and the overlap between the light and the heavy segments. Serine-14 and tyrosine-155 were identified as the residues involved in the covalent and allosteric controls of the enzyme, respectively. Residues 108 and 142 were identified as the cysteine residues reported to be involved in the aggregation of subunits.

Allosteric Regulation↗
PubMed1978

Amino acid sequence of two cyanogen bromide fragments of glycogen phosphorylase.

This communication presents the strategy and experimental details to prove the amino acid sequence of two large fragments of rabbit muscle glycogen phosphorylase generated by cleavage with cyanogen bromide. These fragments, CB18 and CB15, represent 241 of the 841 residues in the whole molecule. In addition to applying methods of automated liquid phase Edman degradation, techniques of selective immobilization and solid phase Edman degradation are used. One of the two cyanogen bromide fragments (CB15) contains two of the sites of cleavage with hydroxylamine which have proved to be important in the overall strategy of determining the complete sequence of this molecule. Together with the accompanying reports by Koide, A., et al., and Titani, K., et al. ((1978) Biochemistry 17 (first and third papers, respectively, in a series in this issue)), the present communication completes the proof of the amino acid sequence of phosphorylase and provides the basis for examining the relationship between its structure and function.

Amino Acid Sequence↗
PubMed1978

Sequence of the carboxyl-terminal 492 residues of rabbit muscle glycogen phosphorylase including the pyridoxal 5'-phosphate binding site.

This communication presents the strategy and experimental details which establish the amino acid sequence of the carboxyl-terminal 492 residues (residues 350 through 841) of rabbit muscle glycogen phosphorylase (EC 2.4.1.1). The heavy segment (Hs), derived from the native enzyme by limited proteolysis with subtilisin, was cleaved with cyanogen bromide to yield 15 fragments. The amino acid sequences of 12 of these are described herein. The sequence of 3 other fragments (CB17C, CB18, and CB15) is described in accompanying reports by Koide, A., et al., and Hermann, j., et al. ((1978) Biochemistry 17 (first and second papers, respectively, in a series in this issue)). These 15 fragments were aligned by analysis of three others generated by cleavage of the heavy segment Hs at asparaginylglycine bonds with hydroxylamine and of four more generated by acid cleavage of aspartylproline bonds. Lysine-679 was identified as the binding site of the essential cofactor pyridoxal 5'-phosphate. These data, together with those reported in the accompanying papers (vide supra), establish the complete sequence of the 841 amino acid residues in glycogen phosphorylase. They provide a chemical basis on which the relationship between structure and function of the enzyme can be examined.

Amino Acid Sequence↗
PubMed1976

Chinese hamster purine-nucleoside phosphorylase: purification, structural, and catalytic properties.

Purine-nucleoside phosphorylase (EC 2.4.2.1; purine-nucleosideorthophosphate ribosyltransferase) was purified to apparent homogeneity from Chinese hamster liver and kidneys and from V79 tissue culture cells. The enzymes from both sources appear to have identical structural and catalytic properties. A simple rapid radioisotope assay capable of detecting 0.1 nmol of product for both directions of the purine-nucleoside phosphorylase reaction is described using Bio-Rad Cu2+ Chelex in Pasteur pipet columns. At 37 degrees C the purified enzyme converts 60 mumol of guanine to guanosine per min per mg of protein. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels indicates that the enzyme is composed of identical subunits of 30 000 molecular weight. The native enzyme behaves as a mixture of dimers of 68 000 molecular weight and trimers of 89 000 molecular weight during Sephadex G-100 chromatography. Sucrose gradient centrifugation indicates that the enzyme had a sedimentation coefficient of 5.4 S, which corresponds to a molecular weight of 94 000 and suggests a trimer structure. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants of 20 muM both hypoxanthine and guanine, 35 muM form guanosine, 50 muM for inosine, and 200 muM for both ribose 1-phosphate and phosphate. During isoelectrofocusing, the enzyme forms a single major band at a pI of 5.25.

Animals↗
PubMed1975

Kinetics of glycogen phosphorylase a with a series of semisynthetic, branched saccharides. A model for binding of polysaccharide substrates.

The requirement of muscle phosphorylase for branched polysaccharide substrates was investigated by kinetic studies on semisynthetic branched saccharides. One series of saccharides was prepared from maltoheptose by oxidizing the reducing group to a carboxyl group and coupling this with an amino group of ethylenediamine. The resulting aminooligosaccharide was coupled with p-nitrophenyl esters of mono-, di-, tetra-, and polycarboxylic aicds to produce saccharides containing one, two, four, and approximately 52 maltodextrin chains per molecule. A similar series of saccharides was prepared from a heterogeneous maltodextrin of average chain length 11.7. Kinetic constants were determined for the reaction with phoshorylase a in the direction of chain elongation. Michaelis constants are equilibrium constants for dissociation of saccharide from the enzyme-AMP-glucose-1P-saccharide complex. The Michaelis constants, expressed in terms of the concentration of nonreducing end groups, are independent of maltodextrin chain length but decrease considerably as the number of chains per molecule increases. Maximum velocities do not differ greatly from that for glycogen. Among the synthetic saccharides, only the polymer behaves similarly to glycogen in exhiiting a decreasing reaction rate as the chains are elongated. The kinetic constants are quantitatively consistent with a model in which two chain termini from the same saccharide molecule bind to the phosphorylase molecule simultaniously, Differences in binding between saccharides having different numbers of equally accessible chains are caused solely by statistical factors in the equilibrium. Highly branched substrates bind better because of their greater multiplicity of two end-group pairs.

Animals↗
PubMed2004

Role of histidine-85 in the catalytic mechanism of thymidine phosphorylase as assessed by targeted molecular dynamics simulations and quantum mechanical calculations.

The structural changes taking place in the enzyme thymidine phosphorylase (TPase, also known as PD-ECGF) that are required to achieve catalytic competence upon binding thymidine and phosphate have been simulated by means of targeted molecular dynamics (tMD). The hinge regions were characterized by structural homology comparisons with pyrimidine nucleoside phosphorylase, whose X-ray structure has been solved both in a closed and in an open form. The rearrangement of residues around the substrate that was observed during the tMD trajectory suggested that His-85 could be playing an important role in the catalytic mechanism. A quantum mechanical study of the reaction in the presence of the most relevant active site residues was then performed at the semiempirical level. The results revealed that His-85 could be involved in the protonation of the pyrimidine base at the O2 position to yield the enol tautomer of the base. To establish the role of this oxygen atom in the reaction, ground states, transition states, and final products were studied using higher level ab initio methods starting from both thymidine and 2-thiothymidine as alternative substrates. Comparison of both transition states showed that replacing the oxygen at position 2 of the pyrimidine base by sulfur should accelerate the reaction rate. Consistent with this result, 2-thiothymidine was shown to be a better substrate for TPase than the natural substrate, thymidine. For simulating the final step of the reaction, tMD simulations were used to study domain opening upon product formation considering both the enol and keto tautomers of thymine. Product release from the enzyme was easiest in the simulation that incorporated the keto tautomer of thymine, suggesting that the enol intermediate spontaneously tautomerizes back to the more energetically stable keto form. These results highlight a previously unreported role for His-85 in the catalytic mechanism of TPase and can have important implications for the design of novel TPase inhibitors.

Amino Acid Sequence↗
PubMed2004

Transition state analysis for human and Plasmodium falciparum purine nucleoside phosphorylases.

Recent studies have shown that Plasmodium falciparum is sensitive to a purine salvage block at purine nucleoside phosphorylase (PNP) and that human PNP is a target for T-cell proliferative diseases. Specific tight-binding inhibitors might be designed on the basis of specific PNP transition state structures. Kinetic isotope effects (KIEs) were measured for arsenolysis of inosine catalyzed by P. falciparum and human purine nucleoside phosphorylases. Intrinsic KIEs from [1'-(3)H]-, [2'-(3)H]-, [1'-(14)C]-, [9-(15)N]-, and [5'-(3)H]inosines were 1.184 +/- 0.004, 1.031 +/- 0.004, 1.002 +/- 0.006, 1.029 +/- 0.006, and 1.062 +/- 0.002 for the human enzyme and 1.116 +/- 0.007, 1.036 +/- 0.003, 0.996 +/- 0.006, 1.019 +/- 0.005, and 1.064 +/- 0.003 for P. falciparum PNPs, respectively. Analysis of KIEs indicated a highly dissociative D(N)A(N) (S(N)1) stepwise mechanism with very little leaving group involvement. The near-unity 1'-(14)C KIEs for both human and P. falciparum PNP agree with the theoretical value for a 1'-(14)C equilibrium isotope effect for oxacarbenium ion formation when computed at the B1LYP/6-31G(d) level of theory. The 9-(15)N KIE for human PNP is also in agreement with theory for equilibrium formation of hypoxanthine and oxacarbenium ion at this level of theory. The 9-(15)N KIE for P. falciparum PNP shows a constrained vibrational environment around N9 at the transition state. A relatively small beta-secondary 2'-(3)H KIE for both enzymes indicates a 3'-endo conformation for ribose and relatively weak hyperconjugation at the transition state. The large 5'-(3)H KIE reveals substantial distortion at the 5'-hydroxymethyl group which causes loosening of the C5'-H5' bonds during the reaction coordinate.

Adenosine Triphosphate↗
PubMed1996

Phosphonate and alpha-fluorophosphonate analogue probes of the ionization state of pyridoxal 5'-phosphate (PLP) in glycogen phosphorylase.

To investigate the role of the essential cofactor pyridoxal phosphate in rabbit muscle glycogen phosphorylase catalysis, two phosphonate analogues of pyridoxal phosphate, 5'-deoxypyridoxal 5'-methylenephosphonic acid and 5'-deoxypyridoxal 5'-difluoromethylenephosphonic acid, have been prepared and reconstituted into apophosphorylase b. UV/Vis spectroscopic and 31P and 19F NMR studies confirmed the successful reconstitution and revealed significant changes in phosphate environment upon nucleotide activation. Both such reconstituted enzymes had activities of approximately 25%-30% of that observed in the native enzyme, while K(m) values were similar to those of the native enzyme. Very similar dependences upon pH of Vmax, K(m), and Vmax/K(m) were found for the two reconstituted enzyme derivatives and the native enzyme despite the considerable difference in phosphonic acid pKa values. These results suggest that pyridoxal phosphate does not function as an essential acid/base catalyst in glycogen phosphorylase; rather they suggest that the cofactor phosphate moiety remain dianionic throughout catalysis and functions as an essential dianion. Mechanistic implications of these findings are discussed.

Animals↗