Snake venom toxins. The amino-acid sequence of a short-neurotoxin homologue from Dendroaspis polylepis polylepis (black mamba) venom

The conformation of 5'-nucleotides in the active site of glycogen phosphorylase b has been deduced from linewidth measurements of protons H-1', H-8 and H-2. It is shown by selective deuteration of the purine ring in position 8 that the orientation of the base is anti in the case of strong activators like AMP and syn in that of weak activators like IMP. The orientation correlation time of the nucleotides in the active site is nearly that of the enzyme, i.e. 160 ns at 21 degrees C.     

The structure of glycogen phosphorylase b with an alkyldihydropyridine-dicarboxylic acid compound, a novel and potent inhibitor

BACKGROUND: In muscle and liver, glycogen concentrations are regulated by the reciprocal activities of glycogen phosphorylase (GP) and glycogen synthase. An alkyl-dihydropyridine-dicarboxylic acid has been found to be a potent inhibitor of GP, and as such has potential to contribute to the regulation of glycogen metabolism in the non-insulin-dependent diabetes diseased state. The inhibitor has no structural similarity to the natural regulators of GP. We have carried out structural studies in order to elucidate the mechanism of inhibition. RESULTS: Kinetic studies with rabbit muscle glycogen phosphorylase b (GPb) show that the compound (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5, 6-tricarboxylate (Bay W1807) has a Ki = 1.6 nM and is a competitive inhibitor with respect to AMP. The structure of the cocrystallised GPb-W1807 complex has been determined at 100K to 2.3 A resolution and refined to an R factor of 0.198 (Rfree = 0.287). W1807 binds at the GPb allosteric effector site, the site which binds AMP, glucose-6-phosphate and a number of other phosphorylated ligands, and induces conformational changes that are characteristic of those observed with the naturally occurring allosteric inhibitor, glucose-6-phosphate. The dihydropyridine-5,6-dicarboxylate groups mimic the phosphate group of ligands that bind to the allosteric site and contact three arginine residues. CONCLUSIONS: The high affinity of W1807 for GP appears to arise from the numerous nonpolar interactions made between the ligand and the protein. Its potency as an inhibitor results from the induced conformational changes that lock the enzyme in a conformation known as the T' state. Allosteric enzymes, such as GP, offer a new strategy for structure-based drug design in which the allosteric site can be exploited. The results reported here may have important implications in the design of new therapeutic compounds.     

Regulation of beta-adrenergic receptors by guanyl-5'-yl imidodiphosphate and other purine nucleotides

Guanyl-5'-yl imidodiphosphate (Gpp(NH)p), GTP, and other purine nucleotides selectively decrease the binding affinity of the beta-adrenergic receptors of frog erythrocyte membranes for beta-adrenergic agonists but not antagonists. Shifts in binding affinity were assessed by determining the ability of unlabeled ligands to compete with (-)-[3H]dihydroalprenolol for the membrane-bound receptors. The magnitude of the"right" shift in the binding displacement curve for any of 13 ligands tested was directly related to the intrinsic activity (maximal stimulatory capacity) of that agent for stimulation of the frog erythrocyte membrane adenylate cyclase. Thus, Gpp(NH)p-induced shifts in binding affinity were greatest for full agonists such as isoproterenol, intermediate for partial agonists such as soterenol, and no shifts were observed for antagonists such as propranolol. Shifts in binding affinity were observed only in preparations where agonist binding to the receptors leads to "coupling" of the receptors with adenylate cyclase. In solubilized preparations where the beta-adrenergic receptors and adenylate cyclase are functionally "uncoupled", Gpp(NH)p did not cause right shifts in agonist receptor binding displacement curves. In particulate preparations the Km of Gpp(NH)p for stimulation of adenylate cyclase was identical with that for its effect on beta-adrenergic agonist binding affinity, 1 to 2 muM. Moreover, the ability of several other nucleotides to cause shifts in receptor binding affinity directly paralleled their previously determined affinities for the nucleotide regulatory sites on adenylate cyclase. Gpp(NH)p also shifted agonist dose-response curves for stimulation of adenylate cyclase, but to the left. As with the effects on the receptor binding curves, the effects of Gpp(NH)p on the "apparent affinities" of agonists for enzyme stimulation were directly related to their intrinsic activities. Gpp(NH)p also markedly increased the intrinsic activity of partial agonists. These results appear to indicate that conformational alterations in adenylate cyclase caused by occupation of nucleotide regulatory sites by Gpp(NH)p are capable of inducing alterations in the beta-adrenergic receptors. These receptor alterations are induced only when the receptors are "coupled" to the enzyme by virtue of agonist binding. The nucleotide-altered conformation of the beta-adrenergic receptors is characterized by decreased binding affinity for agonist but increased functional efficacy in stimulating the enzyme.     

Short-term treatment with prostaglandin E1 analogue has long-term preventive effects on intimal thickening in balloon-injured rat carotid arteries

Based on the absorbance change of indicators with the concentration of hydrogen ion released from an enzyme-catalyzed reaction, a convenient colorimetric method was established for the assay of acidic phospholipase A2 and glycogen phosphorylase b. Brilliant yellow and bromothymol blue were chosen as indicators for assays of acidic phospholipase A2 and glycogen phosphorylase b by following the absorbance changes at 495 and 615 nm, respectively. The method is simple, sample-saving, sensitive and valid for a wide range of enzyme concentrations. It can be extended for assaying other enzymes catalyzing reactions with hydrogen ion concentration changes.     

Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites

Two calcium-stimulated protein kinase activities (ATP:protein phosphotransferase, EC 2.7.1.37) that phosphorylate protein I, a specific synaptic protein, have been identified in homogenates of rat brain. One of these is found in both the particulate and cytosolic fractions and phosphorylates a region of protein I that is phosphorylated in intact synaptosomes in response to calcium but not to cyclic AMP. The stimulation by calcium of the particulate enzyme and of the partially purified cytosolic enzyme requires the addition of calmodulin. It is not yet known whether the particulate and cytosolic enzymes are related. A second calcium-stimulated protein I kinase is found only in the cytosol and phosphorylates a region of protein I that is phosphorylated in intact synaptosomes in response to either calcium or cyclic AMP. The calcium stimulation of this latter kinase is probably mediated by calmodulin, judging from its inhibition by low concentrations of trifluoperazine. Both of the calcium-stimulated protein I kinases are more highly concentrated in brain than in other tissues. The two cytosolic kinases are distinguishable from each other and from myosin light chain kinase and phosphorylase b kinase by their substrate specificities and their chromatographic behavior on DEAE-cellulose.     

Possible arrangement of the five domains in human complement factor I as determined by a combination of X-ray and neutron scattering and homology modeling

Human factor I is a multidomain plasma serine protease with one factor I-membrane attack complex (FIMAC) domain, one CD5 domain, two low-density lipoprotein receptor (LDLr) domains, and one serine protease (SP) domain and is essential for the regulation of complement. The domain arrangement in factor I was determined by X-ray and neutron scattering on serum-derived human factor I (sFI) and recombinant insect cell factor I (rFI). While the radii of gyration of both were the same at 4.05 nm and both had overall lengths of 14 nm, the cross-sectional radii of gyration were different at 1.70 nm for sFI and 1.57 nm for rFI. This difference was attributed to their different means of glycosylation which is complex-type for sFI and high-mannose-type for rFI. Homology models were constructed for the FIMAC, LDLr, and SP domains of factor I using related crystal structures, and CD5 was represented as a globular protein by referencing its electron microscopy dimensions. In these models, 38 of the 40 Cys residues in factor I were predicted to form internal disulfide bridges. The two remaining Cys residues at the N terminus of the FIMAC domain and at the center of the first LDLr domain were potentially not bridged. It was postulated that, if these two Cys residues were bridged to each other, the FIMAC, CD5, and LDLr-1 domains would form a compact triangular arrangement. This hypothesis was tested by automated scattering curve fit searches based on 9600 bilobal models, setting the FIMAC, CD5, and LDLr-1 domains as one lobe and the large SP domain as the other lobe. The searches gave a single small family of similar structures with a separation of 5.9 nm between the centers of the lobes which gave similar good X-ray and neutron fits for both sFI and rFI, despite the different glycosylations of sFI and rFI. These best-fit structures for factor I showed that this domain model is plausible, and suggested that the SP and the CD5 and LDLr-1 domains may present exposed surfaces in factor I whose roles are to interact separately with its substrates C3b and C4b and with cofactor proteins.     

A study of subunit interface residues of fructose-1,6-bisphosphatase by site-directed mutagenesis: effects on AMP and Mg2+ affinities

The structural transformation of fructose-1,6-bisphosphatase upon binding of the allosteric regulator AMP dramatically changes the interactions across the C1-C4 (C2-C3) subunit interface of the enzyme. Asn9, Met18, and Ser87 residues were modified by site-directed mutagenesis to probe the function of the interface residues in porcine liver fructose-1,6-bisphosphatase. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism (CD) spectrometry. No discernible alterations in structure were observed among the wild-type and Asn9Asp, Met18Ile, Met18Arg, and Ser87Ala mutant forms of the enzyme as measured by CD spectrometry. Kinetic analyses revealed 1.6- and 1.8-fold increases in kcat with Met18Arg and Asn9Asp, respectively. The K(m) for fructose 1,6-bisphosphate increased about 2-approximately 4-fold relative to that of the wild-type enzyme in the four mutants. A 50-fold lower Ka value for Mg2+ compared with that of the wild-type enzyme was obtained for Met18Ile with no alteration of the Ki for AMP. However, the replacement of Met18 with Arg caused a dramatic decrease in AMP affinity (20 000-fold) without a change in Mg2+ affinity. Increases of 6- and 2-fold in the Ki values for AMP were found with Asn9Asp and Ser87Ala, respectively. There was no difference in the cooperativity for AMP inhibition between the wild-type and the mutant forms of fructose-1,6-bisphosphatase. This study demonstrates that the mutation of residues in the C1-C4 (C2-C3) interface of fructose-1,6-bisphosphatase can significantly affect the affinity for Mg2+, which is presumably bound 30 A away. Moreover the mutations alternatively reduce AMP and Mg2+ affinities, and this finding may be associated with the destabilization of the corresponding allosteric states of the enzyme. The kinetics and structural modeling studies of the interface residues provide new insights into the conformational equilibrium of fructose-1,6-bisphosphatase.     

AMP analogs: their function in the activation of glycogen phosphorylase b

A series of AMP analogs has been selected in order to better understand the structural requirements (a) for the efficient binding of the activator molecule at the correct site on phosphorylase b from rabbit skeletal muscle and (b) for the activation which is observed. Two types of activation are known, according to Black and Wang [J. Biol. Chem. 243, 5892-5898 (1968)]: either a cooperative response with respect to the activator concentration (like the one which is obtained for AMP itself) or a non-cooperative response observed in the case of IMP. It is shown that the 5'-phosphate moiety is absolutely required for the analog to bind at the correct site (adenine or adenosine bind at another enzymic site), and that the free enthalpy, delta G, corresponding to the association process varies in a complex manner with respect to the substitution of the different positions of the AMP molecule. Moreover, the differences delta G (analog) - delta G (AMP) = delta G obtained for two types of substitution separately do not add up to the same energy difference as the one obtained when the two substitutions are made simultaneously on the AMP molecule. It appears that all the mononucleotides which have been tested up to now may be divided into two classes. Class I (AMP class) is characterized, apart from a strong activation, by the following features: (a) one molecule of analog expels two molecules of bound glucose 6-phosphate as it binds on the enzyme; (b) bound analog protects slowly one crucial cysteinyl residue against attack by 5,5'-dithio-bis(2-nitrobenzoic acid) at 4 degrees C; (c) association of two molecules of dimer is strengthened at 4 degrees C in the presence of the analog. Class II (IMP class) is associated with a weak activation and with the following set of properties: (a) a single molecule of bound glucose 6-phosphate is released as the first molecule of analog binds on the dimer; (b) two slowly reacting cysteinyl residues per subunit are immediately protected against 5,5'-dithio-bis(2-nitrobenzoic acid) by the binding of the analog at 4 degrees C; (c) the analog dissociates the low amount of tetramer which is present at 4 degrees C in the absence of AMP into two molecules of dimer. These results are discussed according to a plausible scheme of transconformations taking place in glycogen phosphorylase b, a model which has been derived earlier by relaxation studies.     

The state of ADP or ATP fixed to the mitochondria by bongkrekate

The reported studies are intended to clarify the binding state of ADP fixed to mitochondria under the influence of bongkrekate, and thus to discern between the affinity increase and reorientation mechanism proposed for the bongkrekate effect. (a) The composition of the intramitochondrial adenine nucleotide pool is not changed under the influence of bongkrekate with and without added nucleotides. (b) The added ADP and ATP fixed by bongkrekate can be identified as AMP, ADP and ATP in the same proportions as in the endogenous pool. (c) The bound nucleotides respond to oxidative phosphorylation or uncoupler stimulated dephosphorylation similar as endogenous nucleotides. It can be concluded that the ADP or ATP fixed under the influence of bongkrekate to the mitochondria are equilibrated with the intramitochondrial adenine nucleotide pool and are active in intramitochondrial phosphate transfer reactions. The results disagree with the affinity increase mechanism but support the reorientation mechanism which postulates that ADP and ATP are trapped in the mitochondria under the influence of bongkrekate in the same amount as there are carrier sites available outside before bongkrekate addition.     

The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition

The structure of a truncated form of the gamma-subunit of phosphorylase kinase (PHKgammat) has been solved in a ternary complex with a non-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti-parallel beta-sheet with the kinase activation segment, the region which in other kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide at a fixed distance from the gamma-phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate. The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognition of dimeric glycogen phosphorylase b is considered.     

The effect of PGE2 and PGF2alpha on the response of guinea pig myometrium to adenine nucleotides in vitro

Prostaglandins E2 and F2alpha potentiate contractile effect induced by adenine nucleotides ATP, ADP and AMP in guinea pig myometrium in vitro. Prostaglandins and nucleotides were added to the organ bath in minute concentrations which have been proved ineffective or slightly contractile when both groups of substances were administered separately. The data of the present work, together with our previously published studies (9, 10, 13), where the action of exogenous adenine nucleotides, NAD and adenosine on rabbit's jejunum in vitro has been proved antagonistic to the contractile effect of various prostaglandins, suggest that prostaglandins and adenine nucleotides appear to block selectively or augment each other's action on various organs. The initial hypothesis that there is a regulatory correlation between endogenous prostaglandins and the function of purinergic nerves also is reinforced.     

Hybrid enzyme of liver phosphorylase and phosphorylase I

A hybrid enzyme (LI) of liver phosphorylase [EC 2.4.1.1.] (L) and phosphorylase I, which is mainly located in brain, was isolated and its enzymatic and immunological properties were examined. The following results were obtained: (1) AMP stimulated the b forms of the hybrid (LIb), I(Ib), and L(Lb); (2) in the presence of AMP, SO42- stimulated Lb more than LIb and inhibited Ib; (3) in the absence of AMP, SO42- stimulated all three isozymes in the order: Ibeta less than LI less than Lbeta; (4) on conversion to the a forms, the activities of L, LI, and I increased 35.5-fold, 3-fold, and 1.2-fold, respectively; (5) the relative inhibition potencies of anti-Lb antibody with LIa and LIb were 63% and 4%, respectively of that with La, and those of anti-Ib antibody with LIa and LIb were 42% and 88%, respectively of that with Ia. Since the ratios of the specific activities of purified La and Ia and of Lbeta and Ibeta are 70: 82 and 2 : 70, respectively (Schliselfeld, 1973), the present findings suggest a 1 : 1 association of I and L subunits in the hybrid molecule.     

Spectroscopic properties of Escherichia coli UDP-N-acetylenolpyruvylglucosamine reductase

Purified uridine diphosphate N-acetylenolpyruvylglucosamine reductase (E.C. 1.1.1.158) was analyzed by circular dichroism (CD) and UV-visible spectroscopy to establish the spectral properties of its tightly bound flavin adenine dinucleotide (FAD) cofactor. The polypeptide backbone displayed a single circular dichroic minimum at 208 nm and a single maximum at 193 nm. The CD spectrum of bound flavin exhibited a single major negative Cotton peak at 364 nm and two minor negative Cotton peaks at 464 and 495 nm. The protein was reversibly unfolded in 9.8 M urea and refolded in buffer in the presence of excess FAD. The refolded enzyme incorporated FAD and catalyzed full activity. The bound FAD displayed an absorption maximum at 464 nm with an extinction coefficient of epsilon 464 = 11700 M-1 cm-1. Anaerobic reduction with dithionite was complete at 1 equiv. Anaerobic reduction with nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), also was essentially complete at 1 equiv and produced a long-wavelength absorbance band characteristic of an FAD-pyridine nucleotide charge transfer complex. Photochemical bleaching in the presence of ethylenediaminetetraacetic acid (EDTA) followed exponential kinetics. None of the anaerobic reductive titrations produced a spectral intermediate characteristic of a flavin semiquinone, and all reduced enzyme species could be fully reoxidized by oxygen, with full recovery of catalytic activity. Photochemically reduced enzyme was reoxidized by titration with either NADP+ or uridine diphospho N-acetylglucosamine enolpyruvate (UNAGEP). Reoxidation by NADP+ reached a chemical equilibrium, whereas reoxidation by UNAGEP was stoichiometric. Binding of NADP+ or UNAGEP to the oxidized form of the enzyme produced a dead-end complex that could be titrated by following a 10-nm red shift in the absorption spectrum of the bound FAD. The Kd of NADP+ for oxidized enzyme was 0.7 +/- 0.3 microM and the Kd of UNAGEP was 2.7 +/- 0.3 microM. Solvent deuterium isotope effects on binding were observed for both NADP+ and UNAGEP, depending on the pH. At pH 8.5, the HKd/DKd was 2.2 for NADP+ and 3.9 for UNAGEP. No spectral changes were observed in the presence of a 40-fold excess of uridine diphospho N-acetylmuramic acid (UNAM) either aerobically or anaerobically. These studies have identified spectral signals for five steps in the kinetic mechanism, have indicated that product formation is essentially irreversible, and have indicated that hydrogen bonding or protonation contributes significantly to ground-state complex formation with the physiological substrate.     

Structure and functions of CD23

This review summarizes recent data on CD23, a low affinity receptor for IgE (Fc epsilon RII). CD23 is the only FcR which does not belong to the immunoglobulin gene superfamily. The CD23 molecule was discovered independently as an IgE receptor on human lymphoblastoid B cells [1], as a cell surface marker expressed on Epstein-Barr-Virus-transformed B cells (EBVCS) [2] and as a B-cell activation antigen (Blast 2) [3]. CD23 was shown to be a low affinity receptor for IgE [4,5]. Similar to most FcR, soluble forms of CD23 (sCD23) are released into extracellular fluids. The soluble fragments formed by proteolytic cleavage of surface CD23 are not only capable of binding IgE (IgE binding factors) but also exhibit multiple functions that are not IgE related. These observations together with the finding that CD23 displays significant homology with Ca(2+)-dependent (C-type) animal lectins, suggested the existence of natural ligands other than IgE. The recent finding that CD23 interacts with CD21, CD11b and CD11c indicates that CD23 should be viewed not only as a low affinity IgE receptor but also as an adhesion molecule involved in cell-cell interaction. After a brief overview of the molecular structure, there follows a discussion of the biological activities ascribed to human CD23.     

Stimulation of glycogenolysis by beta adrenergic agonists in skeletal muscle of mice with the phosphorylase kinase deficiency mutation (I strain)

The mechanism by which beta adrenergic agonist stimulate glycogenolysis in intact skeletal muscle was investigated in mice with the phosphorylase kinase deficiency mutation (I strain). Although extracts of I strain diaphragm muscle had only 3.7% of the phosphorylase kinase activity found in extracts of the control strain (C57BL), incubation of I strain hemidiaphragms in Krebs-Ringer bicarbonate buffer with either isoproterenol or epinephrine resulted in a stimulation of the rate of glycogenolysis. In C57BL diaphragms, the EC50 values for isoproterenol and epinephrine were 2 and 14 nM, respectively. With I strain diaphragms, dl-isoproterenol or l-epinephrine stimulated glycogenolysis as a linear function of the log of the drug concentration with no apparent plateau of response up to concentrations of 30 to 40 mugM. For each 10-fold increase in drug concentration, isoproterenol and epinephrine stimulated glycogenolysis in I strain muscles an additional 0.37 to 0.42 mg/g/hr, a slope in the concentration-response relationship of 0.17 and 0.37, respectively, of that measured in C57BL diaphragms at concentrations around the EC50. The highest glycogenolytic response measured in I strain hemidiaphragms (at 40 mugM isoproterenol) was 80% of the maximal catecholamine-stimulated glycogenolysis in C57BL diaphragms. Both 4 nM and 4 mugM isoproterenol, in a concentration-dependent manner, stimulated phosphorylase b to a conversion in I and C57BL diaphragms and increased cyclic adenosine 3':5'-monophosphate (cyclic AMP) concentrations. The glycogenolytic response to 10.1 nM dl-isoproterenol in both I and C57BL diaphragms was blocked by 34 nM l-propranolol but not by 34 nM d-propranolol. The response to 4 mugM isoproterenol was enhanced by the cyclic nucleotide phosphodiesterase inhibitors papaverine (27 mugM) or dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724, 3 mugM). From the results of these studies, we conclude: 1) Catecholamines stimulate glycogenolysis in skeletal muscle of I mice, as in C57BL mice, by interacting with the beta adrenergic receptor, thereby increasing tissue cyclic AMP concentrations and stimulating phosphorylase b to a conversion. 2) alternative hypotheses for the mechanism of the catecholamine-stimulated decrease in glycogen concentration in I skeletal muscle-inhibition of glycogen synthesis, hyposia and 5'-AMP stimulation of phosphorylase b activity-have been ruled out. 3) the activity of the mutant phosphorylase kinase, although it is only 3.7% of that in extracts of C57BL muscle, is sufficient to produce phosphorylase b to a conversion and thereby account for the glycogenolytic response of I strain muscle to catecholamines.     

Effect of adrenal steroids on preproneurokinin-A gene expression in discrete regions of the rat brain

Glycogen phosphorylase b has been purified to homogeneity from the fat body of larval Manduca sexta. The purification procedure involved ammonium sulfate precipitation, and chromatography of DEAE-cellulose, 5'-AMP-Sepharose and Q-Sepharose. The final product, which showed a single band on SDS-PAGE with a M(r) = 92,500, was purified 50-fold from the original homogenate in a yield of about 3%. The molecular mass of the native purified phosphorylase b was estimated to be 186,000 Da from gel filtration, suggesting that the native enzyme is a dimer. The apparent Km values for glycogen, phosphate and 5'-AMP were 1.4 mM, 82 mM and 1.1 mM, respectively. The enzyme had a pH optimum of 7.05, and was inhibited by ATP, ADP and glucose, but not by trehalose, even at high concentration. Conversion of phosphorylase b into the a form was achieved by incubation with rabbit phosphorylase kinase and Mg(2+)-ATP. The molecular mass of phosphorylase a was estimated to be 250,000 Da by gel filtration chromatography. The specific activity of the a form in the presence of 5'-AMP was 1.6-1.7-fold higher than the specific activity of the b form under the same conditions. Thus, 5'-AMP activates the a form by about 20%, whereas ATP has no effect on the phosphorylase a activity.     

Circular dichroism and electron microscopy of a core Y61F mutant of the F1 gene 5 single-stranded DNA-binding protein and theoretical analysis of CD spectra of four Tyr --> Phe substitutions

A core Y61F mutant of the gene 5 single-stranded DNA-binding protein (g5p) of f1 bacterial virus aggregated when expressed from a plasmid, but, after refolding in vitro, it behaved much like wild-type and may be a stability or folding mutant. Circular dichroism (CD) titrations showed the same cooperative polynucleotide binding modes for Y61F and wild-type g5p. There are n = 4 and n congruent with 2.5 modes for binding to poly[d(A)] at low ionic strengths, but n = 4, n = 3, and n congruent with 2-2.5 modes for binding to fd single-stranded viral DNA (fd ssDNA), where n is the number of nucleotides occluded by each bound g5p monomer in a given mode. Y61F g5p has slightly reduced affinity in the n = 4 mode. Electron microscopy showed that Y61F g5p forms left-handed nucleoprotein superhelices indistinguishable from wild-type. Progression from binding to fd ssDNA in the n = 4 to n = 3 to n congruent with 2-2.5 mode is accompanied by an increase in the number of helical turns, an increase from (7.7 +/- 0.3) to (9.5 +/- 0.3) to ( approximately 10-13) g5p dimers per turn, and a decrease in the number of DNA nucleotides per turn. From CD spectra for four of five possible Y --> F g5p mutants, we infer that the fifth tyrosine, Tyr 56, contributes strongly to the CD. Retention of a strong 229 nm CD band in all mutants indicates that all retain elements of the native structure. Spectra of Y26F, Y34F, and Y61F g5p imply limited mobility of the replacement Phe. Comparison of measured with calculated CD spectra also suggests limited mobility for Tyr 26 and Tyr 34 in g5p in solution, and provides new information that the g5p structure in solution may be dominated by Tyr 41 rotamers differing from that stabilized in the crystal.     

Calf spleen purine nucleoside phosphorylase complexed with substrates and substrate analogues

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway, which provides an alternative to the de novo pathway for the biosynthesis of purine nucleotides. PNP catalyzes the reversible phosphorolysis of 2'-deoxypurine ribonucleosides to the free bases and 2-deoxyribose 1-phosphate. Absence of PNP activity in humans is associated with specific T-cell immune suppression. Its key role in these two processes has made PNP an important drug design target. We have investigated the structural details of the PNP-catalyzed reaction by determining the structures of bovine PNP complexes with various substrates and substrate analogues. The preparation of phosphate-free crystals of PNP has allowed us to analyze several novel complexes, including the ternary complex of PNP, purine base, and ribose 1-phosphate and of the completely unbound PNP. These results provide an atomic view for the catalytic mechanism for PNP proposed by M. D. Erion et al. [(1997) Biochemistry 36, 11735-11748], in which an oxocarbenium intermediate is stabilized by phosphate and the negative charge on the purine base is stabilized by active site residues. The bovine PNP structure reveals several new details of substrate and inhibitor binding, including two phosphate-induced conformational changes involving residues 33-36 and 56-69 and a previously undetected role for His64 in phosphate binding. In addition, a well-ordered water molecule is found in the PNP active site when purine base or nucleoside is also present. In contrast to human PNP, only one phosphate binding site was observed. Although binary complexes were observed for nucleoside, purine base, or phosphate, ribose 1-phosphate binding occurs only in the presence of purine base.     

Major changes in the kinetic mechanism of AMP inhibition and AMP cooperativity attend the mutation of Arg49 in fructose-1,6-bisphosphatase

The significance of subunit interface residues Arg49 and Lys50 in the function of porcine liver fructose-1,6-bisphosphatase was explored by site-directed mutagenesis, initial rate kinetics, and circular dichroism spectroscopy. The Lys50 --> Met mutant had kinetic properties similar to the wild-type enzyme but was more thermostable. Mutants Arg49 --> Leu, Arg49 --> Asp, Arg49 --> Cys were less thermostable than the wild-type enzyme yet exhibited wild-type values for kcat and Km. The Ki for the competitive inhibitor fructose 2,6-bisphosphate increased 3- and 5-fold in Arg49 --> Leu and Arg49 --> Asp, respectively. The Ka for Mg2+ increased 4-8-fold for the Arg49 mutants, with no alteration in the cooperativity of Mg2+ binding. Position 49 mutants had 4-10-fold lower AMP affinity. Most significantly, the mechanism of AMP inhibition with respect to fructose 1,6-bisphosphate changed from noncompetitive (wild-type enzyme) to competitive (Arg49 --> Leu and Arg49 --> Asp mutants) and to uncompetitive (Arg49 --> Cys mutant). In addition, AMP cooperativity was absent in the Arg49 mutants. The R and T-state circular dichroism spectra of the position 49 mutants were identical and superimposable on only the R-state spectrum of the wild-type enzyme. Changes from noncompetitive to competitive inhibition by AMP can be accommodated within the framework of a steady-state Random Bi Bi mechanism. The appearance of uncompetitive inhibition, however, suggests that a more complex mechanism may be necessary to account for the kinetic properties of the enzyme.     

Interaction of ethidium bromide with DNA: effect of LiCl and ethylene glycol

The interaction of the intercalating dye ethidium bromide with several native and synthetic polydeoxyribonucleic acids has been studied by means of circular dichroic spectra. The CD of DNA-ethidium bromide complexes in the 290-360 nm region is characterized, especially at high salt and at high ethylene glycol content, by positive and negative bands near 308 nm and 295 nm, respectively. These dye associated CD bands are unaffected by the addition of LiCl or ethylene glycol, suggesting that the relative conformation of dye and neighboring base pairs does not change when the conformation of the rest of the DNA changes.