Purine nucleoside phosphorylase. Inosine hydrolysis, tight binding of the hypoxanthine intermediate, and third-the-sites reactivity.

Purine nucleoside phosphorylase from calf spleen is a trimer which catalyzes the hydrolysis of inosine to hypoxanthine and ribose in the absence of inorganic phosphate. The reaction occurs with a turnover number of 1.3 x 10(-4) s-1 per catalytic site. Hydrolysis of enzyme-bound inosine occurs at a rate of 2.0 x 10(-3) s-1 to form a stable enzyme-hypoxanthine complex and free ribose. The enzyme hydrolyzes guanosine; however, a tightly-bound guanine complex could not be isolated. The complex with hypoxanthine is stable to gel filtration but can be dissociated by acid, base, or mild denaturing agents. Following gel filtration, the E.hypoxanthine complex dissociates at a rate of 1.9 x 10(-6) s-1 at 4 degrees C and 1.3 x 10(-4) s-1 at 30 degrees C. The dissociation constant for the tightly-bound complex of enzyme-hypoxanthine is estimated to be 1.3 x 10(-12) M at 30 degrees C on the basis of the dissociation rate. The stoichiometry of the reaction is 1 mol of hypoxanthine bound per trimer. The reaction is reversible since the same complex can be formed from enzyme and hypoxanthine. Addition of ribose 1-phosphate to the complex results in the formation of inosine without release of hypoxanthine. Thus, the complex is catalytically competent. Inorganic phosphate or arsenate prevents formation of the tightly-bound E.hypoxanthine complex from inosine or hypoxanthine. Direct binding studies with hypoxanthine in the presence of phosphate result in 3 mol of hypoxanthine bound per trimer with a dissociation constant of 1.6 microM. In the absence of phosphate, three hypoxanthines are bound, but higher hypoxanthine concentrations cause the release of two of the hypoxanthines with an apparent inhibition constant of 130 microM. The results establish that enzymatic contacts with the nucleoside alone are sufficient to destabilize the N-glycosidic bond. In the absence of phosphate, water attacks slowly, causing net hydrolysis. The hydrolytic reaction leaves hypoxanthine stranded at the catalytic site, tightly bound to the enzyme with a conformation related to the transition state. In the phosphorolysis reaction, ribose 1-phosphate causes relaxation of this conformation and rapid release of hypoxanthine.     

Calf spleen purine nucleoside phosphorylase: complex kinetic mechanism,hydrolysis of 7-methylguanosine,and oligomeric state in solution

The active enzyme form was found to be a homotrimer, no active monomers were observed. Only in the presence of an extremely high orthophosphate concentration (0.5 M) or at a low enzyme concentration (0.2 microg/ml) with no ligands present a small fraction of the enzyme is probably in a dissociated and/or non-active form. The specific activity is invariant over a broad enzyme concentration range (0.017 microg/ml-0.29 mg/ml). At concentrations below 0.9 microg/ml and in the absence of ligands the enzyme tends to loose its catalytic activity, while in the presence of any substrate or at higher concentrations it was found to be active as a trimer. In the absence of phosphate the enzyme catalyses the hydrolysis of 7-methylguanosine (m7Guo) with a catalytic rate constant 1.3x10(-3) x s(-1) as compared with the rate of 38 s(-1) for the phosphorolysis of this nucleoside. The initial pre-steady-state phase of the phosphorolysis of m7Guo, 70 s(-1), is almost twice faster than the steady-state rate and indicates that the rate-limiting step is subsequent to the glycosidic bond cleavage. Complex kinetic behaviour with substrates of phosphorolytic direction (various nucleosides and orthophosphate) was observed; data for phosphate as the variable substrate with inosine and guanosine, but not with their 7-methyl counterparts, might be interpreted as two binding sites with different affinities, or as a negative cooperativity. However, the titration of the enzyme intrinsic fluorescence with 0.2 microM-30 mM phosphate is consistent with only one dissociation constant for phosphate, K(d)=220+/-120 microM. Protective effects of ligands on the thermal inactivation of the enzyme indicate that all substrates of the phosphorolytic and the synthetic reactions are able to form binary complexes with the calf spleen purine nucleoside phosphorylase. The purine bases, guanine and hypoxanthine, bind strongly with dissociation constants of about 0.1 microM, while all other ligands studied, including 7-methylguanine and 7-methylhypoxanthine, bind at least 3 orders of magnitude less potently. Binding of guanine and hypoxanthine is about 10-fold weakened by the presence of phosphate. These observations are best interpretable by the complex kinetic mechanism of the phosphorolytic reaction involving (i) random substrate binding, (ii) unusually slow, hence strongly rate-limiting, dissociation of the products guanine and hypoxanthine, but not 7-methylguanine and 7-methylhypoxanthine, and (iii) dual function of the phosphate binding site with phosphate acting as a substrate and as a modifier helping in the release of a purine base after glycosidic bond cleavage.     

Purine nucleoside phosphorylase of rabbit liver. Mechanism of catalysis.

Initial velocity studies and product inhibition patterns for purine nucleoside phosphorylase from rabbit liver were examined in order to determine the predominant catalytic mechanism for the synthetic (forward) and phosphorolytic (reverse) reactions of the enzyme. Initial velocity studies in the absence of products gave intersecting or converging linear double reciprocal plots of the kinetic data for both the synthetic and phosphorolytic reactions of the enzyme. The observed kinetic pattern was consistent with a sequential mechanism, requiring that both substrates add to the enzyme before products may be released. The product inhibition patterns showed mutual competitive inhibition between guanine and guanosine as variable substrates and inhibitors. Ribose 1-phosphate and inorganic orthophosphate were also mutually competitive toward each other. Other combinations of substrates and products gave noncompetitive inhibition. Apparent inhibition constants calculated for guanine as competitive inhibitor and for ribose 1-phosphate as noncompetitive inhibitor of the enzyme, with guanosine as variable substrate, did not vary significantly with increasing concentrations of inorganic orthophosphate as fixed substrate. These results suggest that the mechanism was order and that substrates add to the enzyme in an obligatory order. Dead end inhibition studies carried out in the presence of the products guanine and ribose 1-phosphate, respectively, showed that the kinetically significant abortive ternary complexes of enzyme-guanine-inorganic orthophosphate (EQB) and enzyme-guanose-ribose 1-phosphate (EAP) are formed. The results of dead end inhibition studies are consistent with an obligatory order of substrate addition to the enzyme. The nucleoside or purine is probably the first substrate to form a binary complex with the enzyme, and with which inorganic orthophosphate or ribose 1-phosphate may interact as secondary substrates. The evidences presented in this investigation support an Ordered Theorell-Chance mechanism for the enzyme.     

Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization.

Rabbit liver purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase EC 2.4.2.1.) was purified to homogeneity by column chromatography and ammonium sulfate fractionation. Homogeneity was established by disc gel electrophoresis in presence and absence of sodium dodecyl sulfate, and isoelectric focusing. Molecular weights of 46,000 and 39,000 were determined, respectively, by gel filtration and by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Product inhibition was observed with guanine and hypoxanthine as strong competitive inhibitors for the enzymatic phosphorolysis of guanosine. Respective Kis calculated were 1.25 x 10(-5) M for guanine and 2.5 x 10(-5) M for hypoxanthine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition with guanosine as variable substrate, and an inhibition constant of 3.61 x 10(-4) M was calculated. The protection of essential --SH groups on the enzyme, by 2-mercaptoethanol or dithiothreitol, was necessary for the maintenance of enzyme activity. Noncompetitive inhibition was observed for p-chloromercuribenzoate with an inhibition constant of 5.68 x 10(-6)M. Complete reversal of this inhibition by an excess of 2-mercaptoethanol or dithiothreitol was demonstrated. In the presence of methylene blue, the enzyme showed a high sensitivity to photooxidation and a dependence of photoinactivation on pH, strongly implicating histidine as the susceptible group at the active site of the enzyme. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 5.5 and pH 8.5 The chemical and kinetic evidences suggest that histidine and cysteine may be essential for catalysis. Inorganic orthophosphate (Km 1.54 x 10(-2) M) was an obligatory anion requirement, and arsenate substituted for phosphate with comparable results. Guanosine (Km 5.00 x 10(-5) M), deoxyguanosine (Km 1.00 x 10(-4)M) and inosine (Km 1.33 x 10(-4)M), were substrates for enzymatic phosphorolysis. Xanthosine was an extremely poor substrate, and adenosine was not phosphorylyzed at 20-fold excess of the homogeneous enzyme. Guanine (Km 1.82 x 10(-5)M),ribose 1-phosphate (Km 1.34 x 10(-4) M) and hypoxanthine were substrates for the reverse reaction, namely, the enzymatic synthesis of nucleosides. The initial velocity studies of the saturation of the enzyme with guanosine, at various fixed concentrations of inorganic orthophosphate, suggest a sequential bireactant catalytic mechanism for the enzyme.     

Effects of acyclovir and its metabolites on purine nucleoside phosphorylase

Acyclovir (9-(2-hydroxyethoxymethyl)guanine), the clinically useful antiherpetic agent, is an "acyclic" analogue of 2'-deoxyguanosine. Purine nucleoside phosphorylase partially purified from human erythrocytes did not catalyze detectable phosphorolysis of this drug or any of its metabolites (less than 0.07% of the rate with Guo). However, these compounds were competitive inhibitors of this enzyme with Ino as the variable substrate. Acyclovir per se was a relatively weak inhibitor. Its Ki value (91 microM) was much greater than that for its 8-hydroxy metabolite (Ki = 4.7 microM) but less than that for its carboxylic acid metabolite (9-carboxymethoxy-methylguanine) (K'i = 960 microM). The phosphorylated metabolites of acyclovir were more potent inhibitors than were their guanine nucleotide counterparts. At a phosphate concentration of 50 mM, the apparent Ki values for the mono- (120 microM), di- (0.51 microM), and tri (43 microM)-phosphate esters of acyclovir were 1/2, 1/1200, and 1/26 those for dGMP, dGDP, and dGTP, respectively. The concentration of phosphate did not markedly affect the Ki value of acyclovir but dramatically affected those of its phosphorylated metabolites and their nucleotide counterparts. Decreasing phosphate to a physiological concentration (1 mM) decreased the apparent Ki values for the mono-, di-, and triphosphate esters of acyclovir to 6.6, 0.0087, and 0.31 microM, respectively. Inhibition of the enzyme by acyclovir diphosphate was also influenced by pH. This metabolite of acyclovir is the most potent inhibitor of purine nucleoside phosphorylase reported to date. It has some features of a "multisubstrate" analogue inhibitor.     

The mechanism of inhibition and "reversal" of mitogen-induced lymphocyte activation in a model of purine-nucleoside phosphorylase deficiency

Purine-nucleoside phosphorylase (PNP) is a purine degradative enzyme that catalyzes the phosphorolysis of (deoxy) inosine or (deoxy) guanosine to their respective bases and (deoxy) ribose 1-phosphate. A severe T-cell immune deficiency syndrome with hypouricemia is associated with impaired PNP function. To study the biochemical basis for this syndrome we created an in vitro model of PNP deficiency in mitogen (phytohemagglutinin)-stimulated normal human peripheral blood lymphocytes using guanosine to competitively inhibit deoxyguanosine phosphorolysis. Guanosine-induced guanine toxicity was reversed by adenine. Under these conditions, deoxyguanosine (5-45 microM) diminished mitogen stimulation to 30% of control while increasing the deoxyguanosine triphosphate pool (dGTP) by over 20-fold. Deoxycytidine reversed deoxyguanosine toxicity with a diminution of dGTP accumulation, but no significant change in the deoxycytidine triphosphate pool. Thymidine reversed the deoxyguanosine toxicity, repleted the thymidine triphosphate (dTTP) pool, and caused an even further increase in the accumulation of dGTP. These data support a model of lymphotoxicity in PNP deficiency based on dGTP accumulation with inhibition of ribonucleotide reductase and depletion of the thymidine triphosphate pool. Thymidine triphosphate depletion is reversed by either deoxycytidine or thymidine; however, the former diminishes dGTP accumulation (probably by competition for phosphorylation) and the latter potentiates dGTP accumulation (probably through feedback augmentation of guanosine diphosphate (GDP) reduction by ribonucleotide reductase secondary to an increased dTTP pool).     

Purine nucleoside phosphorylase from Cellulomonas sp.: physicochemical properties and binding of substrates determined by ligand-dependent enhancement of enzyme intrinsic fluorescence,and by protective effects of ligands on thermal inactivation of the enzyme

Purine nucleoside phosphorylase (PNP) from Cellulomonas sp., homotrimeric in the crystalline state, is also a trimer in solution. Other features of the enzyme are typical for "low molecular mass" PNPs, except for its unusual stability at pH 11. Purine bases, alpha-D-ribose-1-phosphate (R1P) and phosphate enhance the intrinsic fluorescence of Cellulomonas PNP, and hence form binary complexes and induce conformational changes of the protein that alter the microenvironment of tryptophan residue(s). The effect due to guanine (Gua) binding is much higher than those caused by other ligands, suggesting that the enzyme preferentially binds a fluorescent, most probably rare tautomeric anionic form of Gua, further shown by comparison of emission properties of the PNP/Gua complex with that of Gua anion and its N-methyl derivatives. Guanosine (Guo) and inosine (Ino) at 100 microM concentration show little and no effect, respectively, on enzyme intrinsic fluorescence, but their protective effect against thermal inactivation of the enzyme points to their forming weak binary complexes with PNP. Binding of Gua, hypoxanthine (Hx) and R1P to the trimeric enzyme is described by one dissociation constant, K(d)=0.46 microM for Gua, 3.0 microM for Hx, and 60 microM for R1P. The binding stoichiometry for Gua (and probably Hx) is three ligand molecules per enzyme trimer. Effects of phosphate on the enzyme intrinsic fluorescence are due not only to binding, but also to an increase in ionic strength, as shown by titration with KCl. When corrected for effects of ionic strength, titration data with phosphate are most consistent with one dissociation constant, K(d)=270 microM, but existence of a very weak binding site with K(d)>50 mM could not be unequivocally ruled out. Binding of Gua to the PNP/phosphate binary complex is weaker (K(d)=1.7 microM) than to the free enzyme (K(d)=0.46 microM), suggesting that phosphate helps release the purine base in the catalytic process of phosphorolysis.The results indicate that nonlinear kinetic plots of initial velocity, typical for PNPs, including Cellulomonas PNP, are not, as generally assumed, due to cooperative interaction between monomers forming the trimer, but to a more complex kinetic mechanism than hitherto considered.     

The dissociation of 1-N6-ethenoadenosine diphosphate from regulated actomyosin subfragment 1

The effective rate of dissociation of 1-N6-ethenoadenosine diphosphate (epsilon ADP) from the regulated actin X subfragment 1 X epsilon ADP complex of rabbit skeletal muscle is approximately 10-15 times smaller in the absence of calcium ion compared to the presence of calcium ion. The decrease in fluorescence emission with dissociation of the bound epsilon ADP fitted two exponential terms. The evidence is consistent with a kinetic scheme in which two first-order transitions precede the dissociation step: (Formula: see text) where D is epsilon ADP, A is regulated actin, M is subfragment 1, the asterisks refer to the degree of fluorescence enhancement, and AM(D) is a collision complex in equilibrium with free epsilon ADP. Both rate constants k-2 and k-1 were reduced approximately 15-fold in the absence of calcium ion. The rate constants for the dissociation of epsilon ATP, epsilon ADP X Pi, formed in the enzyme cycle, and epsilon ADP are all reduced in the absence of calcium ion; consequently, the primary effect in calcium regulation of the actin-subfragment 1 ATPase is on the rate constant of a transition (or transitions) between actomyosin-nucleoside phosphate complexes.     

A calorimetric study of 3d metal ions-acyclovir interactions. The 2-hydroxyethoxymethyl group of acyclovir mimics the role of ribose in deoxy-guanosine and guanosine promoting the coordination through N(7)

The equilibrium constants and enthalpic values of metal acyclovir complexes have been determined by calorimetry for Co(II) (log K=0.96+/-0.05, DeltaH (kJ/mol)=-19.7+/-1.3), Ni(II) (log K=1.39+/-0.03, DeltaH (kJ/mol)=-21.5+/-1.0), Cu(II) (log K=1.83+/-0.03, DeltaH (kJ/mol)=-23.2+/-0.8) and Zn(II) (log K=0.71+/-0.06, DeltaH (kJ/mol)=-18.6+/-1.5). The equilibrium constants are similar to those of the divalent ions with guanosine and 2,9-dimethylpurine. By comparison with previous thermodynamic data, it can be shown that the 2-hydroxyethoxymethyl group promotes coordination through N(7) versus N(1) of the guanine ring for 3d metal ions. These results reveal that the 2-hydroxyethoxymethyl group placed on the purine ring of guanine in acyclovir causes a greater effect than that of the 9-methyl in purines and similar to or greater than that of the ribose moiety in guanosine. The 2-hydroxyethyoxymethyl group of acyclovir mimics the role of ribose in deoxy-guanosine and guanosine promoting a similar coordination chemistry (with very close log K and DeltaH values) for acyclovir, deoxy-guanosine and guanosine with divalent metals.     

The control of pyruvate kinases of Escherichia coli. II. Effectors and regulatory properties of the enzyme activated by ribose 5-phosphate.

The pyruvate kinases of Escherichia coli activated by ribose 5-phosphate (RP) has been partially purified. The active form of the enzyme has a molecular weight of about 180 000 as judged by sucrose density gradient centrifugations and Sephadex G-150 chromatography. On dissociation in the absence of sulfhydryl reagents such as dithiothreitol, the enzyme is inactivated and it has a molecular weight of about 110 000. Various substrates and effectors of the enzyme, with the exception of phosphate, do not influence the association-dissociation equilibrium of the enzyme. The enzyme, unlike pyruvate kinases from many other sources, is not activated by potassium ions. Sulfate and phosphate ions are inhibitory to the enzyme. Phosphate seems to be an allosteric inhibitor and its effect is completely antagonized by activators. The enzyme is activated in an allosteric manner by two classes of compounds, nucleoside monophosphates and sugar phosphates of the hexose monophosphate pathway. Amongst the nucleotides, guanosine 5'-phosphate and adenosine 5'-phosphate are the most effective activators. Amongst the hexose monophosphate pathway intermediates, RP is the most powerful activator, with apparent activation constants as low as 1 Mu. Sugar phosphates esterified at C-1 or both terminal positions are entirely ineffective in activation. The effectors act by changing the Michaelis constant for the substrates. Both of the substrates of the enzyme, adenosine diphosphate and phosphoenolpyruvate, yield cooperative-concentration plots in the presence of unsaturating concentrations of the fixed changing substrate. The initial velocity plots for both substrates become hyperbolic in the presence of saturating concentrations of RP.     

Bovine brain purine-nucleoside phosphorylase purification, characterization, and catalytic mechanism.

Bovine brain purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) was purified to homogeneity at a specific activity of 78 mumol min-1 mg of protein-1. A molecular weight of 78 000-80 000 was calculated for the native enzyme by fel filtration on Sephadex. Gel electrophoresis in the presence of sodium dodecyl sulfate indicated subunits of molecular weight of 38 000. Chemical and kinetic studies strongly implicated histidine and cysteine as catalytic groups at the active site of the enzyme. The pKa's determined for ionizable groups at the active site of the free enzyme were 5.8 and 8.2. Enzyme completely inactivated by p-chloromercuribenzoate was partially reactivated enzyme. A strong susceptibility to photooxidation in presence of methylene blue was observed. Photoinactivation was pH dependent, implicating histidine as the susceptible group at the active site. A rapid loss of catalytic activity upon incubation at 55 degrees C suggested heat lability. An activation energy of 9.6 kcal/mol was calculated. The nature of the catalytic mechanism of the enzyme was investigated, and initial velocity studies showed linear converging patterns of double-reciprocal plots of the data, consistent with a sequential catalytic mechanism. The product inhibition pattern was at variance with both the ordered Bi-Bi and random mechanisms. The observed competition between purine and nucleoside, and between inorganic orthophosphate and ribose 1-phosphate for this ordered mechanism, suggest a Theorell-Chance mechanism. Michaelis constants determined for substrates of the enzyme were 4.35 X 10(-5) M for guanosine, 3.00 X 10(-5) M for guanine, and 2.15 X 10(-2) M for inorganic orthophosphate.     

Reactions of d-glyceraldehyde 3-phosphate dehydrogenase facilitated by oxidized nicotinamide–adenine dinucleotide

Transient kinetic methods have been used to study the influence of NAD(+) on the rate of elementary processes of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase. In the pH range 5-8 NAD(+) is bound to the enzyme during the following elementary processes of the mechanism: phosphorolysis of the acyl-enzyme, its formation from 1,3-diphosphoglycerate and the enzyme and the formation and breakdown of the glyceraldehyde 3-phosphate-enzyme complex. The rates of these four elementary processes only equal or exceed the turnover rate of the enzyme when NAD(+) is bound and are as much as 10(4) times the rates in the absence of NAD(+). Autocatalysis of the reductive dephosphorylation of 1,3-diphosphoglycerate occurs when glyceraldehyde 3-phosphate release is rate determining because NAD(+) is a reaction product. An important feature of the enzyme mechanism is that the negative-free-energy change of a chemical reaction, acyl-enzyme formation, is linked in a simple way to the positive-free-energy change of a dissociation reaction, NAD(+) release.     

Escherichia coli phnN,encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways

An enzymatic pathway for synthesis of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B. Hove-Jensen, J. Bacteriol. 178:714-722, 1996). The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase. The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously. The reaction sequence is ribose 5-phosphate --> ribose 1-phosphate --> ribose 1,5-bisphosphate --> PRPP. Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine + P(i) --> guanine + ribose 1-phosphate. The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (C-P) bond by a C-P lyase. The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end. PhnN was purified almost to homogeneity and characterized. The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor. The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5'-XMP, and by cochromatography of the reaction product with authentic PRPP.     

Substrate specificity and kinetic mechanism of purine nucleoside phosphorylase from Mycobacterium tuberculosis

Purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) is numbered among targets for persistence of the causative agent of tuberculosis. Here, it is shown that MtPNP is more specific to natural 6-oxopurine nucleosides and synthetic compounds, and does not catalyze the phosphorolysis of adenosine. Initial velocity, product inhibition and equilibrium binding data suggest that MtPNP catalyzes 2′-deoxyguanosine (2dGuo) phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate (P_i) binds first followed by 2dGuo, and ribose 1-phosphate dissociates first followed by guanine. pH-rate profiles indicated a general acid as being essential for both catalysis and 2dGuo binding, and that deprotonation of a group abolishes P_i binding. Proton inventory and solvent deuterium isotope effects indicate that a single solvent proton transfer makes a modest contribution to the rate-limiting step. Pre-steady-state kinetic data indicate that product release appears to contribute to the rate-limiting step for MtPNP-catalyzed reaction.     

Trehalose phosphate synthesis in Streptomyces hygroscopicus: purification of guanosine diphosphate D-glucose: D-glucose-6-phosphate 1-glucosyl-transferase

Guanosine diphosphate d-glucose:d-glucose-6-phosphate 1-glucosyl-transferase was purified approximately 100-fold from extracts of Streptomyces hygroscopicus. The purified enzyme catalyzed the transfer of glucose from guanosine diphosphate-d-glucose to glucose-6-phosphate to form trehalose phosphate and guanosine diphosphate. The enzyme was specific for these two substrates and was stimulated by the addition of magnesium ions. The product was characterized as alpha-alpha-trehalose-6-phosphate by its physical and chemical properties. The enzyme was present in a large number of Streptomyces species, suggesting that this group of organisms synthesized trehalose phosphate in a unique manner. This enzyme was not detected in fungi, since these organisms utilized uridine diphosphate-d-glucose as the glucosyl donor.     

Functional groups at the catalytic site of F1 adenosine triphosphatase

The protection of F1 ATPase by inorganic phosphate, ADP, ATP, and magnesium ion against inactivation by 1-fluoro-2,4-dinitrobenzene, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, respectively, has been investigated. Dissociation equilibrium constants and rate constants for the labeling reactions have been deduced from a quantitative treatment of the kinetic data. Comparison of these dissociation constants with each other and with the corresponding literature values indicates that the essential Tyr, Arg, Lys, and Glu or Asp residues are indeed located at the catalytic site of the enzyme. Examination of the rate constants for the labeling reactions in the presence of excess inorganic phosphate, ADP, ATP, or magnesium ion, respectively, suggests that the essential phenol and amino groups are located nearer to the bound inorganic phosphate or the gamma-phosphate group than to the alpha- or beta-phosphate group of the bound ATP, that the essential guanidinium group is located nearer to the alpha- or beta-phosphate group than to the gamma-phosphate group of the bound ATP or the bound inorganic phosphate, and that the essential carboxylate group is located slightly farther away but complexed with magnesium ion which it shares with the bound inorganic phosphate. A mechanism consistent with these topographical relationships is proposed for the catalytic hydrolysis and synthesis of ATP.     

Aspects of the chemistry of d-glyceraldehyde 3-phosphate dehydrogenase

Crystalline d-glyceraldehyde 3-phosphate dehydrogenase from lobster tail contains 4 moles of NAD(+) bound and reacts specifically with 4 moles of iodoacetic acid/mole of tetramer. The essential thiol group of d-glyceraldehyde 3-phosphate dehydrogenase appears to react with iodoacetic acid with a rate constant for the overall process that is independent of the extent of carboxymethylation. The d-glyceraldehyde 3-phosphate dehydrogenase-NAD(+) absorption band has a variable molar extinction coefficient in the presence of phosphate that may be correlated with a proton dissociation of pK 6.86. The binding of NAD(+) to d-glyceraldehyde 3-phosphate dehydrogenase weakens as alkylating agents react with the enzyme, and NAD(+) promotes the reactivity of the essential thiol group. It is suggested that, on binding to d-glyceraldehyde 3-phosphate dehydrogenase, NAD(+) lowers the pK of the essential thiol group, resulting in a catalytic role of NAD(+) in the reaction catalysed by d-glyceraldehyde 3-phosphate dehydrogenase. If this theory is correct, then it is likely that a proton will be liberated during the phosphorolysis of the acyl-enzyme rather than in the redox step.     

Shift in nucleotide conformational equilibrium contributes to increased rate of catalysis of GpAp versus GpA in barnase

The microbial ribonuclease barnase exhibits low catalytic activity toward GpN dinucleotides, where G is guanosine, p is phosphate and N represents any nucleoside. When a phosphate is added to the 3'-end, as in GpNp, substrate affinity is enhanced by one order of magnitude, and the catalytic rate by two. In order to gain insight into this phenomenon, we analyzed the nucleotide conformations and protein-nucleotide interactions of 4 ns molecular dynamics (MD) trajectories of complexes of barnase with guanylyl(3'-5') adenosine (GpA) and guanylyl(3'-5') adenosine 3'-monophosphate (GpAp), respectively, in the presence of solvent and counter ions. We found that, in a majority of the bound GpA conformations, the guanine base was firmly bound to the recognition site. The phosphate and adenosine moieties pointed into the solvent, and interactions with key catalytic residues were absent. In contrast, the bound GpAp adopted conformations in which all of the nucleotide portions remained tightly bound to the enzyme and interactions with key catalytic residues were maintained. These observations indicate that, for GpA, a significant proportion of the bound nucleotide adopts non-productive conformations and that adding the terminal phosphate as in GpAp shifts the equilibrium of the bound conformations towards structures capable of undergoing catalysis. Incorporating this property into the kinetic equations yields an increase in both the apparent rate constant (kcat) and the apparent dissociation constant (K(M)) for GpAp versus GpA. The increase in K(M), caused by the presence of additional non-productive binding modes for GpA, should however be counterbalanced by the propensity of free GpA to adopt folded conformations in solution, which are unable to bind the enzyme and by the tighter binding of GpAp (Giraldo J, Wodak SJ, Van Belle D. Conformational analysis of GpA and GpAp in aqueous solution by molecular dynamics and statistical methods. J Mol Biol 1998; 283:863-882). Addition of the terminal phosphate is shown to significantly influence the collective motion of the enzyme in a manner that fosters interactions with key catalytic residues, representing a further likely contribution to the catalytic rate enhancement.     

Coenzyme binding by triphosphopyridine nucleotide dependent isocitrate dehydrogenase from beef liver. Equilibrium and kinetics studies.

The binding of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP) dependent isocitrate dehydrogenase from beef liver cytoplasm was studied by several equilibrium techniques (ultracentrifugation, molecular sieving, ultrafiltration, fluorescence). Two binding sites (per dimeric enzyme molecule) were found with slightly different dissociation constants (0.5 and 0.12 muM) and fluorescence yields (7.7 and 6.3). A ternary complex was formed between enzyme, isocitrate, and NADPH, in which NADPH dissociation constant was 5 muM. On the contrary, no binding of NADPH to the enzyme took place in the presence of magnesium isocitrate. Dialysis experiments showed the existence of 1 NADP binding site/dimer, with a dissociation constant of 26 muM. When NADPH was present with the enzyme in the proportion of 1 molecule/dimer, the dissociation constant of NADP was decreased fourfold, reaching a value quantitatively comparable to the Michaelis constant. The kinetics of coenzyme binding was followed using the stopped-flow technique with fluorescence detection. NADPH binding to the enzyme occurred through one fast reaction (k1 = 20 muM-1 s-1). Dissociation of NADPH took place upon NADP binding; however, equilibrium as well as kinetic data were incompatible with a simple competition scheme. Dissociation of NADPH from the enzyme upon magnesium isocitrate binding was preceded by the formation of a transitory ternary complex in which the fluorescence of NADPH was only about 30% of that in the enzyme-NADPH complex. Then interaction between the conenzymes and the involvement of ternary complexes in the catalytic mechanism are discussed in relation with what is known about the regulatory role of the coenzyme (Carlier, M. F., and Pantaloni, D. (1976), Biochemistry, 15, 1761-1766).     

Structural basis for substrate specificity of Escherichia coli purine nucleoside phosphorylase

Purine nucleoside phosphorylase catalyzes reversible phosphorolysis of purine nucleosides and 2'-deoxypurine nucleosides to the free base and ribose (or 2'-deoxyribose) 1-phosphate. Whereas the human enzyme is specific for 6-oxopurine ribonucleosides, the Escherichia coli enzyme accepts additional substrates including 6-oxopurine ribonucleosides, 6-aminopurine ribonucleosides, and to a lesser extent purine arabinosides. These differences have been exploited in a potential suicide gene therapy treatment for solid tumors. In an effort to optimize this suicide gene therapy approach, we have determined the three-dimensional structure of the E. coli enzyme in complex with 10 nucleoside analogs and correlated the structures with kinetic measurements and computer modeling. These studies explain the preference of the enzyme for ribose sugars, show increased flexibility for active site residues Asp204 and Arg24, and suggest that interactions involving the 1- and 6-positions of the purine and the 4'- and 5'-positions of the ribose provide the best opportunities to increase prodrug specificity and enzyme efficiency.