Trimeric purine nucleoside phosphorylase: Exploring postulated one-third-of-the-sites binding in the transition state

Transition-state analogue inhibitors, immucillins, were reported to bind to trimeric purine nucleoside phosphorylase (PNP) with the stoichiometry of one molecule per enzyme trimer [Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm, V. L. Biochem. 1998, 37, 8615]. In attempts to observe and better understand the nature of this phenomenon we have conducted calorimetric titrations of the recombinant calf PNP complexed with immucillin H. However, by striking contrast to the earlier reports, we have not observed negative cooperativity and we got the stoichiometry of three immucillin molecules per enzyme trimer. Similar results were obtained from fluorimetric titrations, and for other inhibitors bearing features of the transition state. However, we observed apparent cooperativity between enzyme subunits and apparent lower stoichiometry when we used the recombinant enzyme not fully purified from hypoxanthine, which is moped from Escherichia coli cells. Results presented here prove that one-third-of-the-sites binding does not occur for trimeric PNP, and give the highly probable explanation why previous experiments were interpreted in terms of this phenomenon.     

Kinetic properties of Cellulomonas sp. purine nucleoside phosphorylase with typical and non-typical substrates: implications for the reaction mechanism

Phosphorolysis catalyzed by Cellulomonas sp. PNP with typical nucleoside substrate, inosine (Ino), and non-typical 7-methylguanosine (m7Guo), with either nucleoside or phosphate (Pd) as the varied substrate, kinetics of the reverse synthetic reaction with guanine (Gua) and ribose-1-phosphate (R1P) as the varied substrates, and product inhibition patterns of synthetic and phosphorolytic reaction pathways were studied by steady-state kinetic methods. It is concluded that, like for mammalian trimeric PNP, complex kinetic characteristics observed for Cellulomonas enzyme results from simultaneous occurrence of three phenomena. These are sequential but random, not ordered binding of substrates, tight binding of one substrate purine bases, leading to the circumstances that for such substrates (products) rapid-equilibrium assumptions do not hold, and a dual role of Pi, a substrate, and also a reaction modifier that helps to release a tightly bound purine base.     

Crystal structure of calf spleen purine nucleoside phosphorylase with two full trimers in the asymmetric unit: important implications for the mechanism of catalysis

The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining specificity of trimeric PNPs and is in line with the proposed mechanism of catalysis in which this contact helps to stabilize the negative charge that accumulates on O(6) of the purine base in the transition state. In the present crystal structure the loop between Thr60 and Ala65 was found in a different conformation than that observed in crystal structures of trimeric PNPs up to now. Due to this change a new wide entrance is opened into the active site pocket, which is otherwise buried in the interior of the protein. Hence, our present crystal structure provides no obvious indication for obligatory binding of one of the substrates before binding of a second one; it is rather consistent with random binding of substrates. All these results provide new data for clarifying the mechanism of catalysis and give reasons for the non-Michaelis kinetics of trimeric PNPs.     

KINETIC PROPERTIES OF CELLULOMONAS SP. PURINE NUCLEOSIDE PHOSPHORYLASE WITH TYPICAL AND NON-TYPICAL SUBSTRATES: IMPLICATIONS FOR THE REACTION MECHANISM

Phosphorolysis catalyzed by Cellulomonas sp. PNP with typical nucleoside substrate, inosine (Ino), and non-typical 7-methylguanosine (m⁷Guo), with either nucleoside or phosphate (P_i) as the varied substrate, kinetics of the reverse synthetic reaction with guanine (Gua) and ribose-1-phosphate (R1P) as the varied substrates, and product inhibition patterns of synthetic and phosphorolytic reaction pathways were studied by steady-state kinetic methods. It is concluded that, like for mammalian trimeric PNP, complex kinetic characteristics observed for Cellulomonas enzyme results from simultaneous occurrence of three phenomena. These are sequential but random, not ordered binding of substrates, tight binding of one substrate purine bases, leading to the circumstances that for such substrates (products) rapid-equilibrium assumptions do not hold, and a dual role of P_i, a substrate, and also a reaction modifier that helps to release a tightly bound purine base.     

Fluorescence studies of calf spleen purine nucleoside phosphorylase (PNP) complexes with guanine and 9-deazaguanine

Interactions of trimeric calf spleen purine nucleoside phosphorylase (PNP) with guanine (Gua) and its analogue, 9-deazaguanine (9-deaza-Gua), were studied by means of the steady-state fluorescence. The aim was to test the hypothesis that the enzyme stabilizes the anionic form of purine, inferred previously from the unusual increase of fluorescence observed after binding of guanine by calf spleen PNP. We have found that the dissociation constants obtained form titration experiments are in fact pH-independent in the range 7.0-10.25 for both PNP/Gua and PNP/9-deaza-Gua complexes. In particular, at pH 7.0 we found Kd = 0.12 +/- 0.02 micro M for Gua and 0.16 +/- 0.01 micro M for 9-deaza-Gua, while at the conditions where there is more than 40% of the anionic form the respective values were Kd = 0.15 +/- 0.01 micro M for Gua (pH 9.0) and 0.25 +/- 0.02 micro M for 9-deaza-Gua (pH 10.25). Hence, the enzyme does not prefer binding of anionic forms of these ligands in respect to the neutral ones. This result questions the involvement of the anionic forms in the reaction catalyzed by trimeric PNPs, and contradicts the hypothesis of a strong hydrogen bond formation between the enzyme Asn 243 residue and the purine N7 position.     

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.     

Fluorescence studies of calf spleen purine nucleoside phosphorylase (PNP) complexes with guanine and 9-deazaguanine

Interactions of trimeric calf spleen purine nucleoside phosphorylase (PNP) with guanine (Gua) and its analogue, 9-deazaguanine (9-deaza-Gua), were studied by means of the steady-state fluorescence. The aim was to test the hypothesis that the enzyme stabilizes the anionic form of purine, inferred previously from the unusual increase of fluorescence observed after binding of guanine by calf spleen PNP. We have found that the dissociation constants obtained form titration experiments are in fact pH-independent in the range 7.0-10.25 for both PNP/Gua and PNP/9-deaza-Gua complexes. In particular, at pH 7.0 we found K _d = 0.12 ± 0.02 μ M for Gua and 0.16 ± 0.01 μ M for 9-deaza-Gua, while at the conditions where there is more than 40% of the anionic form the respective values were K _d = 0.15 ± 0.01 μ M for Gua (pH 9.0) and 0.25 ± 0.02 μ M for 9-deaza-Gua (pH 10.25). Hence, the enzyme does not prefer binding of anionic forms of these ligands in respect to the neutral ones. This result questions the involvement of the anionic forms in the reaction catalyzed by trimeric PNPs, and contradicts the hypothesis of a strong hydrogen bond formation between the enzyme Asn 243 residue and the purine N(7) position.     

Towards the mechanism of trimeric purine nucleoside phosphorylases: stopped-flow studies of binding of multisubstrate analogue inhibitor - 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine

The binding of multisubstrate analogue inhibitor - 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine (PME-6-thio-Gua) to purine nucleoside phosphorylase from Cellulomonas sp. at 20 degrees C, in 20 mM Hepes buffer with ionic strength adjusted to 50 mM using KCl, at several pH values between 6.5 and 8.2, was investigated using a stopped-flow spectrofluorimeter. The kinetic transients registered after mixing a protein solution with ligand solutions of different concentrations were simultaneously fitted by several association reaction models using nonlinear least-squares procedure based on numerical integration of the chemical kinetic equations appropriate for given model. It is concluded that binding of a PME-6-thio-Gua molecule by each of the binding sites is sufficiently well described by one-step process, with a model assuming interacting binding sites being more probable than a model assuming independent sites. The association rate constants derived from experimental data, assuming one step binding and independent sites, are decreasing with an increase in pH, changing from 30 to 6 microM(-1)s(-1) per binding site. The dissociation rate constants are in the range of 1-3 s(-1), and they are rather insensitive of changes in pH. Interestingly, for each pH value, the one-step binding model with interacting sites results in the association rate constant per site 1.5-4 times smaller for the binding of the first ligand molecule than that for the binding of the second one. Decrease of association constants with pH indicate that the enzyme does not prefer binding of the naturally occurring anionic form of the 6-thioguanine ring (pK(a) 8.7) resulting from a dissociation of N(1)-H. This finding supports the mechanism in which hydrogen bond interaction of N(1)-H with Glu204 (Glu 201 in mammalian PNPs) is crucial in the catalytic process. Results obtained also indicate that, in contrast to transition-state analogues, for which binding is followed by a conformational change, binding of multisubstrate analogue inhibitors to trimeric PNPs is a one-step process.     

Cloning, expression, purification, and some properties of calf purine nucleoside phosphorylase

Calf spleen purine nucleoside phosphorylase (PNP) is considered a model enzyme for the trimeric PNPs subfamily. PCR amplification of the calf phosphorylase from the calf spleen library, cloning, overexpression of the recombinant PNP, its enzymatic activity and interactions with typical ligands of mammalian wild type PNP are described. Relative activity of the recombinant phosphorylase versus several substrates is similar to the respective values obtained for the enzyme isolated from calf spleen. As for the nonrecombinant calf PNP, the unusual fluorescence properties of the PNP/guanine complex were observed and characterized.     

Cloning,Expression, Purification,and Some Properties of Calf Purine Nucleoside Phosphorylase

Calf spleen purine nucleoside phosphorylase (PNP) is considered a model enzyme for the trimeric PNPs subfamily. PCR amplification of the calf phosphorylase from the calf spleen library, cloning, overexpression of the recombinant PNP, its enzymatic activity and interactions with typical ligands of mammalian wild type PNP are described. Relative activity of the recombinant phosphorylase versus several substrates is similar to the respective values obtained for the enzyme isolated from calf spleen. As for the nonrecombinant calf PNP, the unusual fluorescence properties of the PNP/guanine complex were observed and characterized.     

Crystal structure of the purine nucleoside phosphorylase (PNP) from Cellulomonas sp. and its implication for the mechanism of trimeric PNPs

The three-dimensional structure of the trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp. has been determined by X-ray crystallography. The binary complex of the enzyme with orthophosphate was crystallized in the orthorhombic space group P212121 with unit cell dimensions a=64.1 A, b=108.9 A, c=119.3 A and an enzymatically active trimer in the asymmetric unit. X-ray data were collected at 4 degrees C using synchrotron radiation (EMBL/DESY, Hamburg). The structure was solved by molecular replacement, with the calf spleen PNP structure as a model, and refined at 2.2 A resolution. The ternary "dead-end" complex of the enzyme with orthophosphate and 8-iodoguanine was obtained by soaking crystals of the binary orthophosphate complex with the very weak substrate 8-iodoguanosine. Data were collected at 100 K with CuKalpha radiation, and the three-dimensional structure refined at 2.4 A resolution. Although the sequence of the Cellulomonas PNP shares only 33 % identity with the calf spleen enzyme, and almost no identity with the hexameric Escherichia coli PNP, all three enzymes have many common structural features, viz. the nine-stranded central beta-sheet, the positions of the active centres, and the geometrical arrangement of the ligands in the active centres. Some similarities of the surrounding helices also prevail. In Cellulomonas PNP, each of the three active centres per trimer is occupied by orthophosphate, and by orthophosphate and base, respectively, and small structural differences between monomers A, B and C are observed. This supports cooperativity between subunits (non-identity of binding sites) rather than existence of more than one binding site per monomer, as previously suggested for binding of phosphate by mammalian PNPs. The phosphate binding site is located between two conserved beta- and gamma-turns and consists of Ser46, Arg103, His105, Gly135 and Ser223, and one or two water molecules. The guanine base is recognized by a zig-zag pattern of possible hydrogen bonds, as follows: guanine N-1...Glu204 O(epsilon1)...guanine NH2...Glu204 O(epsilon2). The exocyclic O6 of the base is bridged via a water molecule to Asn246 N(delta), which accounts for the inhibitory, but lack of substrate, activity of adenosine. An alternative molecular mechanism for catalysis by trimeric PNPs is proposed, in which the key catalytic role is played by Glu204 (Glu201 in the calf and human enzymes), while Asn246 (Asn243 in the mammalian enzymes) supports binding of 6-oxopurines rather than catalysis. This mechanism, in contrast to that previously suggested, is consistent with the excellent substrate properties of N-7 substituted nucleosides, the specificity of trimeric PNPs versus 6-oxopurine nucleosides and the reported kinetic properties of Glu201/Ala and Asn243/Ala point variants of human PNP.     

Stopped-flow studies of guanine binding by calf spleen purine nucleoside phosphorylase

The binding of guanine to calf spleen purine nucleoside phosphorylase at 20 degrees C, in 20 mM Hepes-NaOH buffer, pH 7.0, at several ionic strength between 5 and 150 mM was investigated using a stopped-flow spectrofluorimeter. The kinetic transients registered after mixing a protein solution with ligand solutions of different concentrations were simultaneously fitted by several association reaction models using nonlinear least-squares procedure based on numerical integration of the chemical kinetic equations appropriate for given model. It is concluded that binding of a guanine molecule by each of the binding sites is a two-step process and that symmetrical trimeric calf spleen purine nucleoside phosphorylase represents a system of (identical) interacting binding sites. The interaction is visible through relations between the rate constants and non-additivity of changes in "molar" fluorescence for different forms of PNP-guanine complexes. It is also probable that electrostatic effects in guanine binding are weak, which indicates that it is the neutral form of the ligand which is bound and dissociated by PNP molecule.     

Interactions of calf spleen purine nucleoside phosphorylase with 8-azaguanine, and a bisubstrate analogue inhibitor: implications for the reaction mechanism

Interactions of calf spleen purine nucleoside phosphorylase (PNP) with a non-typical substrate, 8-azaguanine (8-azaG), and a bisubstrate analogue inhibitor, 9-(2-phosphonylmethoxyethyl)-8-azaguanine (PME-azaG), were investigated by means of steady-state fluorescence spectroscopy. Both 8-azaG and PME-azaG form fluorescent complexes with the enzyme, and dissociation constants are comparable to the appropriate parameters (Km or Ki) obtained from kinetic measurements. PME-azaG inhibits both the phosphorolytic and synthetic pathway of the reaction in a competitive mode. The complex of 8-azaG with PNP is much weaker than the previously reported Gua-PNP complex, and its dissociation constant increases at pH > 7, where 8-azaG exists predominantly as the monoanion (pKa approximately 6.5). The fluorescence difference spectrum of the PNP/8-azaG complex points to participation of the N(7)H or/and N(8)H tautomers of the neutral substrate, and the 9-(2-phosphonylmethoxyethyl) derivative also exists as a neutral species in the complex with PNP. The latter conclusion is based on spectral characteristics of the PNP/PME-azaG complex, confirmed by fluorimetric determination of dissociation constants, which are virtually pH-independent in the range 6-7. These findings testify to involvement of the neutral purine molecule, and not its monoanion, as the substrate in the reverse, synthetic reaction. It is proposed that, in the reverse reaction pathway, the natural purine substrate is bound to the enzyme as the neutral N(7)H tautomer, which is responsible for the reported strong fluorescence of the guanine-PNP complex.     

Interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with the cationic and zwitterionic forms of the fluorescent substrate N(7)-methylguanosine

Steady-state and time-resolved fluorescence spectroscopy, and enzyme kinetics, were applied to study the reaction of purine nucleoside phosphorylase (PNP) from Escherichia coli with its substrate N(7)-methylguanosine (m7Guo), which consists of an equilibrium mixture of cationic and zwitterionic forms (pK(a)=7.0), each with characteristic absorption and fluorescence spectra, over the pH range 6-9, where absorption and intrinsic fluorescence of the enzyme are virtually unchanged. The pH-dependence of kinetic constants for phosphorolysis of m7Guo were studied under condition where the population of the zwitterion varied from 10% to 100%. This demonstrated that, whereas the zwitterion is a 3- to 6-fold poorer substrate, if at all, than the cation for the mammalian enzymes, both ionic species are almost equally good substrates for E. coli PNP. The imidazole-ring-opened form of m7Guo is neither a substrate nor an inhibitor of phosphorolysis. Enzyme fluorescence quenching, and concomitant changes in absorption and fluorescence spectra of the two ionic species of m7Guo on binding, showed that both forms are bound by the enzyme, the affinity of the zwitterion being 3-fold lower than that of the cation. Binding of m7Guo is bimodal, i.e., an increase in ligand concentration leads to a decrease in the association constant of the enzyme-ligand complex, typical for negative cooperativity of enzyme-ligand binding, with a Hill constant <1. This is in striking contrast to interaction of the enzyme with the parent Guo, for which the association constant is independent of concentration. The weakly fluorescent N(7)-methylguanine (m7Gua), the product of phosphorolysis of m7Guo, is a competitive non-substrate inhibitor of phosphorolysis (K(i)=8+/-2 microM) and exhibits negative cooperativity on binding to the enzyme at pH 6.9. Quenching of enzyme emission by the ligands is a static process, inasmuch as the mean excited-state lifetime, =2.7 ns, is unchanged in the presence of the ligands, and the constants K(SV) may therefore be considered as the association constants for the enzyme-ligand complexes. In the pH range 9.5-11 there is an instantaneous reversible decrease in PNP emission of approximately 15%, corresponding to one of the six tyrosine residues per subunit readily accessible to solvent, and OH- ions. Relevance of the overall results to the mechanism of phosphorolysis, and binding of substrates/inhibitors is discussed.     

Formycin A and its N-methyl analogues, specific inhibitors of E. coli purine nucleoside phosphorylase (PNP): induced tautomeric shifts on binding to enzyme, and enzyme-->ligand fluorescence resonance energy transfer

Steady-state and time-resolved emission spectroscopy were used to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitors, viz. formycin B (FB), and formycin A (FA) and its N-methylated analogues, N(1)-methylformycin A (m(1)FA), N(2)-methylformycin A (m(2)FA) and N(6)-methylformycin A (m(6)FA), in the absence and presence of phosphate (P(i)). Complex formation led to marked quenching of enzyme tyrosine intrinsic fluorescence, with concomitant increases in fluorescence of FA and m(6)FA, independently of the presence of P(i). Fluorescence of m(1)FA in the complex increased only in the presence of P(i), while the weak fluorescence of FB appeared unaffected, independently of P(i). Analysis of the emission, excitation and absorption spectra of enzyme-ligand mixtures pointed to fluorescence resonance energy transfer (FRET) from protein tyrosine residue(s) to FA and m(6)FA base moieties, as a major mechanism of protein fluorescence quenching. With the non-inhibitor m(2)FA, fluorescence emission and excitation spectra were purely additive. Effects of enzyme-FA, or enzyme-m(6)FA, interactions on nucleoside excitation and emission spectra revealed shifts in tautomeric equilibria of the bound ligands. With FA, which exists predominantly as the N(1)-H tautomer in solution, the proton N(1)-H is shifted to N(2), independently of the presence of P(i). Complex formation with m(6)FA in the absence of P(i) led to a shift of the amino-imino equilibrium in favor of the imino species, and increased fluorescence at 350 nm; by contrast, in the presence of P(i), the equilibrium was shifted in favor of the amino species, accompanied by higher fluorescence at 430 nm, and a higher affinity for the enzyme, with a dissociation constant K(d)=0.5+/-0.1 microM, two orders of magnitude lower than that for m(6)FA in the absence of P(i) (K(d)=46+/-5 microM). The latter was confirmed by analysis of quenching of enzyme fluorescence according to a modified Stern-Volmer model. Fractional accessibility values (f(a)) varied from 0.31 for m(1)FA to 0.70 for FA, with negative cooperative binding of m(1)FA and FB, and non-cooperative binding of FA and m(6)FA. For all nucleoside ligands, the best model describing binding stoichiometry was one ligand per native enzyme hexamer. Fluorescence decays of PNP, FA and their mixtures were best fitted to a sum of two exponential terms, with average lifetimes () affected by their interactions. Complex formation resulted in a 2-fold increase in of FA, and a 2-fold decrease in of enzyme fluorescence. The amplitude of the long-lifetime component also increased, confirming the shift of the tautomeric equilibrium in favor of the N(2)-H species. The findings have been examined in relation to enzyme-nucleoside binding deduced from structural studies.     

Fluorimetric assay for terminal deoxynucleotidyl transferase activity

A fluorimetric assay for measuring terminal deoxynucleotidyl transferase activity in purified and crude enzyme preparations has been developed. Etheno-substituted deoxynucleotides are shown to be substrates of the enzyme. The assay involves polymerization of the fluorescent nucleotide 1,N6-ethenodeoxyadenosine triphosphate (epsilon dATP) on an oligodeoxynucleotide initiator, [poly(deoxyadenylic acid) with an average chain length of 50 residues] under the reaction conditions used in the standard radiometric assay. The incorporation of epsilon dATP into polymer is quantitated by fluorescence after isolation and nuclease digestion of the product. The enzymological properties of etheno substrates were also determined. Epsilon dATP binds about twofold tighter than dATP to terminal transferase, but has a twofold-lower catalytic rate. However etheno substitution does not affect initiator binding. The fluorimetric assay is suitable for clinical analysis of terminal transferase in human leukemias, and may be a useful adjunct to recently developed immunochemical methods which detect protein, not activity.     

Investigation of ligand binding and protein dynamics in Bacillus subtilis chorismate mutase by transverse relaxation optimized spectroscopy-nuclear magnetic resonance

The structural and dynamical consequences of ligand binding to a monofunctional chorismate mutase from Bacillus subtilis have been investigated by solution NMR spectroscopy. TROSY methods were employed to assign 98% of the backbone (1)H(N), (1)H(alpha), (15)N, (13)C', and (13)C(alpha) resonances as well as 86% of the side chain (13)C resonances of the 44 kDa trimeric enzyme at 20 degrees C. This information was used to map chemical shift perturbations and changes in intramolecular mobility caused by binding of prephenate or a transition state analogue to the X-ray structure. Model-free interpretation of backbone dynamics for the free enzyme and its complexes based on (15)N relaxation data measured at 600 and 900 MHz showed significant structural consolidation of the protein in the presence of a bound ligand. In agreement with earlier structural and biochemical studies, substantial ordering of 10 otherwise highly flexible residues at the C-terminus is particularly notable. The observed changes suggest direct contact between this protein segment and the bound ligand, providing support for the proposal that the C-terminus can serve as a lid for the active site, limiting diffusion into and out of the pocket and possibly imposing conformational control over substrate once bound. Other regions of the protein that experience substantial ligand-induced changes also border the active site or lie along the subunit interfaces, indicating that the enzyme adapts dynamically to ligands by a sort of induced fit mechanism. It is believed that the mutase-catalyzed chorismate-to-prephenate rearrangement is partially encounter controlled, and backbone motions on the millisecond time scale, as seen here, may contribute to the reaction barrier.     

Oligomeric structure of mammalian purine nucleoside phosphorylase in solution determined by analytical ultracentrifugation

The influence of phosphate, ionic strength, temperature and enzyme concentration on the oligomeric structure of calf spleen purine nucleoside phosphorylase (PNP) in solution was studied by analytical ultracentrifugation methods. Sedimentation equilibrium analysis used to directly determine the enzyme molecular mass revealed a trimeric molecule with Mr = (90.6 +/- 2.1) kDa, regardless the conditions investigated: protein concentration in the range 0.02-1.0 mg/ml, presence of up to 100 mM phosphate and up to 200 mM NaCl, temperature in the range 4-25 degrees C. The sedimentation coefficient (6.04 +/- 0.02) S, together with the diffusion coefficient (6.15 +/- 0.11) 10(-7) cm2/s, both values obtained from the classic sedimentation velocity method at 1.0 mg/ml PNP concentration in 20 mM Hepes, pH 7.0, yielded a molecular mass of (90.2 +/- 1.6) kDa as expected for the trimeric enzyme molecule. Moreover, as shown by active enzyme sedimentation, calf spleen PNP remained trimeric even at low protein concentrations (1 microg/ml). Hence in solution, similar like in the crystalline state, calf spleen PNP is a homotrimer and previous suggestions for dissociation of this enzyme into more active monomers, upon dilution of the enzyme or addition of phosphate, are incorrect.     

Escherichia coli purine nucleoside phosphorylase II, the product of the xapA gene

Purine nucleoside phosphorylases (PNPs, E. C. 2.4.2.1) use orthophosphate to cleave the N-glycosidic bond of beta-(deoxy)ribonucleosides to yield alpha-(deoxy)ribose 1-phosphate and the free purine base. Escherichia coli PNP-II, the product of the xapA gene, is similar to trimeric PNPs in sequence, but has been reported to migrate as a hexamer and to accept xanthosine with comparable efficiency to guanosine and inosine, the usual physiological substrates for trimeric PNPs. Here, we present a detailed biochemical characterization and the crystal structure of E.coli PNP-II. In three different crystal forms, PNP-II trimers dimerize, leading to a subunit arrangement that is qualitatively different from the "trimer of dimers" arrangement of conventional high molecular mass PNPs. Crystal structures are compatible with similar binding modes for guanine and xanthine, with a preference for the neutral over the monoanionic form of xanthine. A single amino acid exchange, tyrosine 191 to leucine, is sufficient to convert E.coli PNP-II into an enzyme with the specificity of conventional trimeric PNPs, but the reciprocal mutation in human PNP, valine 195 to tyrosine, does not elicit xanthosine phosphorylase activity in the human enzyme.     

Specific interactions of 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase with coenzymes.

The binding of oxidized and reduced coenzyme (NAD+ and NADH) to 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase has been studied spectrophotometrically and fluorimetrically. The binding of NAD+ to the acylated sturgeon enzyme is characterized by a significant quenching of the enzyme fluorescence (about 25%) and the induction of a difference spectrum in the ultraviolet absorbance region of the enzyme. Both of these spectroscopic properties are quantitatively distinguishable from those of the corresponding binary enzyme-NAD+ complex. Binding isotherms estimated by gel filtration of the acylated enzyme are in close agreement to those obtained by spectrophotometric and fluorimetric titrations. Up to four NAD+ molecules are bound to the enzyme tetramer. No anticooperativity can be detected in the binding of oxidized coenzyme, which is well described on the basis of a single class of four binding sites with a dissociation constant of 25 muM at 10 degrees C, pH 7.0. The binding of NADH to the acylenzyme has been characterized spectrophotometrically. The absorption band of the dihydronicotinamide moiety of the coenzyme is blue-shifted to 335 nm with respect to free NADH. In addition, a large hypochromicity (23%) is observed together with a significant increase of the bandwidth at half height of this absorption band. This last property is specific to the acylenzyme-DADH complex, since it disappears upon arsenolysis of the acylenzyme. The binding affinity of NADH to the acylated enzyme has been estimated by performing simultaneous spectrophotometric and fluorimetric titrations of the NADH appearance upon addition of NAD+ to a mixture of enzyme and excess glyceraldehyde 3-phosphate. In contrast to NAD+, the reduced coenzyme NADH appears to be relatively strongly bound to the acylated enzyme, the dissociation constant of the acylenzyme-NADH complex being estimated as 2.0 muM at 25 degrees C. In addition a large quenching of the NADH fluorescence (about 83%) is observed. The comparison of the dissociation constants of the coenzyme-acylenzyme complexes and the corresponding Michaelis constants suggests a reaction mechanism of the enzyme in which significant formation and dissociation of NAD+-acylenzyme and NADH-acylenzyme complexes occur. Under physiological conditions the activity of the enzyme can be regulated by the ratio of oxidized and reduced coenzymes. Possible reasons for the lack of anticooperativity in coenzyme binding to the acylated form of the enzyme are discussed.