Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transition state structure for ADP-ribosylation of eukaryotic elongation factor 2 catalyzed by diphtheria toxin

Bacterial protein toxins are the most powerful human poisons known, exhibiting an LD(50) of 0.1-1 ng kg(-)(1). A major subset of such toxins is the NAD(+)-dependent ADP-ribosylating exotoxins, which include pertussis, cholera, and diphtheria toxin. Diphtheria toxin catalyzes the ADP ribosylation of the diphthamide residue of eukaryotic elongation factor 2 (eEF-2). The transition state of ADP ribosylation catalyzed by diphtheria toxin has been characterized by measuring a family of kinetic isotope effects using (3)H-, (14)C-, and (15)N-labeled NAD(+) with purified yeast eEF-2. Isotope trapping experiments yield a commitment to catalysis of 0.24 at saturating eEF-2 concentrations, resulting in suppression of the intrinsic isotope effects. Following correction for the commitment factor, intrinsic primary kinetic isotope effects of 1.055 +/- 0.003 and 1.022 +/- 0.004 were observed for [1(N)'-(14)C]- and [1(N)-(15)N]NAD(+), respectively; the double primary isotope effect was 1.066 +/- 0.004 for [1(N)'-(14)C, 1(N)-(15)N]NAD(+). Secondary kinetic isotope effects of 1.194 +/- 0.002, 1.101 +/- 0.003, 1.013 +/- 0.005, and 0.988 +/- 0.002 were determined for [1(N)'-(3)H]-, [2(N)'-(3)H]-, [4(N)'-(3)H]-, and [5(N)'-(3)H]NAD(+), respectively. The transition state structure was modeled using density functional theory (B1LYP/6-31+G) as implemented in Gaussian 98, and theoretical kinetic isotope effects were subsequently calculated using Isoeff 98. Constraints were varied in a systematic manner until the calculated kinetic isotope effects matched the intrinsic isotope effects. The transition state model most consistent with the intrinsic isotope effects is characterized by the substantial loss in bond order of the nicotinamide leaving group (bond order = 0.18, 1.99 A) and weak participation of the attacking imidazole nucleophile (bond order = 0.03, 2.58 A). The transition state structure imparts strong oxacarbenium ion character to the ribose ring even though significant bond order remains to the nicotinamide leaving group. The transition state model presented here is asymmetric and consistent with a dissociative S(N)1 type mechanism in which attack of the diphthamide nucleophile lags behind departure of the nicotinamide.     

Catalytic and allosteric mechanism of AMP nucleosidase from primary, beta-secondary, and multiple heavy atom kinetic isotope effects

Adenosine 5'-phosphate was synthesized with specific heavy atom substitutions to permit measurement of V/K kinetic isotope effects for the N-glycohydrolase activity of the allosteric AMP nucleosidase and the acid-catalyzed solvolysis of these compounds. The effects of allosteric activation on the kinetic isotope effects together with the kinetic mechanism of AMP nucleosidase [DeWolf, W. E., Jr., Emig, F. A., & Schramm, V. L. (1986) Biochemistry 25, 4132-4140] indicate that the kinetic isotope effects are fully expressed. Comparison of individual primary and secondary kinetic isotope effects with combined isotope effects and the isotope effect of the reverse reaction indicated that kinetic isotope effects in AMP nucleosidase arise from a single step in the reaction mechanism. Under these conditions, kinetic isotope effects can be used to interpret transition-state structure for AMP nucleosidase. Changes in kinetic isotope effects occurred as a function of allosteric activator, demonstrating that allosteric activation alters transition-state structure for AMP nucleosidase. Kinetic isotope effects, expressed as [V/K(normal isotope]/[V/K(heavy isotope)], were observed with [2'-2H]AMP (1.061 +/- 0.002), [9-15N]AMP (1.030 +/- 0.003), [1'-2H]AMP (1.045 +/- 0.002), and [1'-14C]AMP (1.035 +/- 0.002) when hydrolyzed by AMP nucleosidase in the absence of MgATP. Addition of MgATP altered the [2'-2H]AMP effect (1.043 +/- 0.002) and the [1'-2H]AMP effect (1.030 +/- 0.003) and caused a smaller decrease of the 14C and 15N effects. Multiple heavy atom substitutions into AMP caused an increase in observed isotope effects to 1.084 +/- 0.004 for [1'-2H,1'-14C]AMP and to 1.058 +/- 0.002 for [9-15N,1'-14C]AMP with the enzyme in the absence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth inhibition by methionine analog inhibitors of S-adenosylmethionine biosynthesis in the absence of polyamine depletion

Four methionine analog inhibitors of methionine adenosyltransferase, the enzyme which catalyzes S-adenosylmethionine biosynthesis, were tested in cultured L1210 cells for their effects on cell growth, leucine incorporation, S-adenosylmethionine (AdoMet) formation and polyamine biosynthesis. The IC50 values were as follows: selenomethionine, 0.13 mM; L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cis-AMB), 0.4 mM; cycloleucine, 5 mM and 2-aminobicyclo[2.1.1]hexane-2-carboxylic acid, 5 mM. At IC50 levels, the analogs significantly reduced AdoMet pools by approximately 50% while not similarly affecting leucine incorporation or polyamine biosynthesis. In combination with inhibitors of polyamine biosynthesis, growth inhibition was greatly increased with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase, but only slightly increased with alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Overall, the data indicate that the methionine analogs, and particularly L-cis-AMB, seem to inhibit cell growth by interference with AdoMet biosynthesis. Since polyamine biosynthesis is not affected, the antiproliferative effect may be mediated through perturbations of certain transmethylation reactions.     

Glutaraldehyde crosslinking analysis of the C12E8 solubilized H,K-ATPase

A soluble porcine H,K-ATPase preparation was obtained with the nonionic detergent, C12E8. ATP hydrolysis by the soluble H,K-ATPase was stimulated with respect to the native preparation at pH 6.1, while the K(+)-phosphatase activity was comparable to the native enzyme. The soluble enzyme demonstrated characteristic ligand-dependent effects on ATP hydrolysis, including ATP activation of K(+)-stimulated hydrolysis with a K0.5 of 28 +/- 4 microM ATP, and inhibition with an IC50 of 2.1 mM ATP. The activation and inhibition of ATP hydrolysis by K+ was also observed with a K0.5 for activation of 2.8 +/- 0.4 mM KCl at 2.0 mM ATP (pH 6.1) and inhibition with an IC50 of 135 mM KCl at 0.05 mM ATP. 2-Methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080), a specific inhibitor of the native H,K-ATPase, competitively inhibited the K(+)-stimulated activity with a Ki of 0.035 microM. The soluble enzyme was stable with a t0.5 for ATPase activity of 6 h between 4 and 11 degrees C. The demonstration of these related ligand responses in the catalytic reactions of the soluble preparation indicates that it is an appropriate medium for investigation of the subunit associations of the functional H,K-ATPase. Subunit associations of the active soluble enzyme were assessed following treatment with the crosslinking reagent, glutaraldehyde. The distribution of crosslinked particles was independent of the soluble protein concentration in the crosslinking buffer within the protein range 0.3 to 2.0 mg/ml or the detergent to protein ratio varied from 1 to 15 (w/w). The crosslinked pattern was unaffected by the presence or absence of K during crosslinking or nucleotide concentration. These observations suggest that crosslinking occurs in associated subunits that do not undergo rapid associations dependent upon enzyme turnover. Phosphorylation of the soluble enzyme with 0.1 mM MgATP produced a phosphoprotein at 94 kDa. A phosphoprotein obtained after glutaraldehyde treatment exhibited identical electrophoretic mobility to the crosslinked particle identified by silver stain. Glutaraldehyde treatment of soluble protein fractions resolved on a linear 10-35% glycerol gradient revealed several smaller peptides partially resolved from the crosslinked pump particle, but no active fraction enriched in the monomeric H,K-ATPase. This data indicates that the functional porcine gastric H,K-ATPase is organized as a structural dimer.     

The conformations of a substrate and a product bound to the active site of S-adenosylmethionine synthetase

S-Adenosylmethionine (AdoMet) is the most widely used alkyl group donor in biological systems. The formation of AdoMet from ATP and L-methionine is catalyzed by S-adenosylmethionine synthetase (AdoMet synthetase). Elucidation of the conformations of enzyme-bound substrates, product, and inhibitors is important for the understanding of the catalytic mechanism of the enzyme and the design of new inhibitors. To obtain structural data for enzyme-bound substrates and product, we have used two-dimensional transferred nuclear Overhauser effect spectroscopy to determine the conformation of enzyme-bound AdoMet and 5'-adenylyl imidodiphosphate (AMPPNP). AMPPNP, an analogue of ATP, is resistant to the ATP hydrolysis activity of AdoMet synthetase because of the presence of a nonhydrolyzable NH-link between the beta- and gamma-phosphates but is a substrate for AdoMet formation during which tripolyphosphate is produced. AdoMet and AMPPNP both bind in an anti conformation about the glycosidic bond. The ribose rings are in C3'-exo and C4'-exo conformations in AdoMet and AMPPNP, respectively. The differences in ribose ring conformations presumably reflect the different steric requirements of the C5' substituents in AMPPNP and AdoMet. The NMR-determined conformations of AdoMet and AMPPNP were docked into the E. coli AdoMet synthetase active site taken from the enzyme.ADP. Pi crystal structure. Since there are no nonexchangeable protons either in the carboxy-terminal end of the methionine segment of AdoMet or in the tripolyphosphate segment of AMPPNP, these portions of the molecules were modeled into the enzyme active site. The interactions of AdoMet and AMPPNP with the enzyme predict the location of the methionine binding site and suggest how the positive charge formed on the sulfur during AdoMet synthesis is stabilized.     

MgATP-induced conformational change of the catalytic subunit of cAMP-dependent protein kinase

Conformational changes of the cAMP-dependent protein kinase (PKA) catalytic (C) subunit are critical for the catalysis of gamma-phosphate transfer from adenosine 5'-triphosphate (ATP) to target proteins. Time-resolved fluorescence anisotropy (TRFA) was used to investigate the respective roles of Mg(2+), ATP, MgATP, and the inhibitor peptide (IP20) in the conformational changes of a 5,6-carboxyfluorescein succinimidyl ester (CF) labeled C subunit ((CF)C). TRFA decays were fit to a biexponential equation incorporating the fast and slow rotational correlation times phi(F) and phi(S). The (CF)C apoenzyme exhibited the rotational correlation times phi(F)=1.8+/-0.3 ns and phi(S)=20.1+/-0.6 ns which were reduced to phi(F)=1.1+/-0.2 ns and phi(S)=13.3+/-0.9 ns in the presence of MgATP. The reduction in rotational correlation times indicated that the (CF)C subunit adopted a more compact shape upon formation of a (CF)C.MgATP binary complex. Neither Mg(2+) (1-3 mM) nor ATP (0.4 mM) alone induced changes in the (CF)C subunit conformation equivalent to those induced by MgATP. The effect of MgATP was removed in the presence of ethylenediaminetetraacetic acid (EDTA). The addition of IP20 and MgATP to form the (CF)C x MgATP x IP20 ternary complex produced rotational correlation times similar to those of the (CF)C x MgATP binary complex. However, IP20 alone did not elicit an equivalent reduction in rotational correlation times. The results indicate that binding of MgATP to the C subunit may induce conformation changes in the C subunit necessary for the proper stereochemical alignment of substrates in the subsequent phosphorylation.     

NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme

Proton NMR was used to study the interaction of beta,gamma-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind MgepsilonATP [Hamada, M., Palmieri, R. H., Russell, G. A., & Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 microM, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 microM]. Time-dependent nuclear Overhauser effects (NOE's) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE's at 250 and 500 MHz. The H2' to H1' distance so obtained (3.07 A) was within the range established by X-ray and model-building studies of nucleotides (2.9 +/- 0.2 A). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (chi = 63 +/- 12 degrees) and 3'-endo/O1'-endo ribose puckers (sigma = 96 +/- 12 degrees). Enzyme- and peptide-bound MgATP molecules exhibited different C4'-C5' torsional angles (gamma) of 170 degrees and 50 degrees, respectively. Ten intermolecular NOE's from protons of the enzyme and four such NOE's from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of beta,gamma-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual consistency of interproton and Cr3+ to proton distances obtained in metal-ATP complexes of both the enzyme and the peptide suggests that the conformation of the peptide is very similar to that of residues 1-45 of the enzyme. When this was assumed to be the case and when molecular models and a computer graphics system were used, MgATP could be fit into the X-ray structure of adenylate kinase in a unique manner such that all of the distances determined by NMR were accommodated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transition state structure of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli and its similarity to transition state analogues

Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes reactions linked to polyamine metabolism, quorum sensing pathways, methylation reactions, and adenine salvage. It is a candidate target for antimicrobial drug design. Kinetic isotope effects (KIEs) were measured on the MTAN-catalyzed hydrolysis of 5'-methylthioadenosine (MTA) to determine the transition state structure. KIEs measured at pH 7.5 were near unity due to the large forward commitment to catalysis. Intrinsic KIEs were expressed by increasing the pH to 8.5. Intrinsic KIEs from MTAs labeled at 1'-(3)H, 1'-(14)C, 2'-(3)H, 4'-(3)H, 5'-(3)H, 9-(15)N, and Me-(3)H(3) were 1.160 +/- 0.004, 1.004 +/- 0.003, 1.044 +/- 0.004, 1.015 +/- 0.002, 1.010 +/- 0.002, 1.018 +/- 0.006, and 1.051 +/- 0.002, respectively. The large 1'-(3)H and small 1'-(14)C KIEs indicate that the Escherichia coli MTAN reaction undergoes a dissociative (D(N)A(N)) (S(N)1) mechanism with little involvement of the leaving group or participation of the attacking nucleophile at the transition state, causing the transition state to have significant ribooxacarbenium ion character. A transition state constrained to match the intrinsic KIEs was located with density functional theory [B3LYP/6-31G(d,p)]. The leaving group (N9) is predicted to be 3.0 A from the anomeric carbon. The small beta-secondary 2'-(3)H KIE of 1.044 corresponds to a modest 3'-endo conformation for ribose and a H1'-C1'-C2'-H2' dihedral angle of 53 degrees at the transition state. Natural bond orbital analysis of the substrate and the transition state suggests that the 4'-(3)H KIE is due to hyperconjugation between the lone pair (n(p)) of O3' and the antibonding (sigma) orbital of the C4'-H4' group, and the methyl-(3)H(3) KIE is due to hyperconjugation between the n(p) of sulfur and the sigma of methyl C-H bonds. Transition state analogues that resemble this transition state structure are powerful inhibitors, and their molecular electrostatic potential maps closely resemble that of the transition state.     

Isolation and characterization of BsuE methyltransferase, a CGCG specific DNA methyltransferase from Bacillus subtilis

The DNA methyltransferase M-BsuE that recognizes the sequence 5'-CGCG-3' has been isolated from Bacillus subtilis strain ISE15. A 1600-fold purification of M-BsuE was achieved by column chromatography on phosphocellulose, heparin-Sepharose, and DEAE-Sepharose. DNA methyltransferase activity was monitored in the column eluants radiochemically by the transfer of tritiated methyl groups from radiolabeled S-adenosylmethionine to poly(dGdC)-poly(dGdC) DNA, a sensitive and specific substrate for M-BsuE activity. The DNA sequence specificity of this methyltransferase activity was confirmed enzymatically by demonstrating that M-BsuE-methylated DNA was selectively protected from cleavage by the restriction enzyme isoschizomers, ThaI and FnuDII. Purified M-BsuE has an apparent molecular size of 41,000-43,000 as determined by gel filtration and migrates as a 41-kDa protein in a sodium dodecyl sulfate-polyacrylamide gel. DNA methylation by M-BsuE is dependent upon the presence of S-adenosylmethionine and 2-mercaptoethanol. M-BsuE methyltransferase activity is optimal at 37 degrees C in the presence of 50 mM Tris-HCl, pH 7.8, 25 mM KCl, 6 microM S-adenosylmethionine, 5 mM 2-mercaptoethanol, and 10 mM EDTA. M-BsuE methylates the external cytidine in its recognition sequence in both linear and supercoiled DNA. A unique property of M-BsuE is its ability to methylate 5'-CGCG-3' in Z-DNA.     

Phospholipid metabolism and lysosomal enzyme secretion by leukocytes. Effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP.

The effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of beta-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3':5'-monophosphate (0.1--1.0 mM) reduced beta-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 MM), but not other nucleotides, was found to potentiate beta-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition the isotope incarparation into these acidic phospholipids by the two mucleotides has not been defined. Dibutyryl cyclic adenosine 3':5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.     

Stable isotope fractionation by Clostridium pasteurianum. 2. Regulation of sulfite reductases by sulfur amino acids and their influence on sulfur isotope fractionation during SO32- and SO42- reduction

In addition to an assimilatory sulfite reductase, studies of cultures of Clostridium pasteurianum supplemented with methionine, cysteine, and 35SO42- provides evidence for another reductase which is induced by SO32-. This inducible reductase appears to be dissimaltory because of the copious sulfide production arising when the cells are grown on SO32-. Cysteine can repress the assimilatory sulfite reductase but does not affect the inducible reductase. During late logarithmic growth on 1 mM SO42- + 10mM cysteine, depression of the inducible reductase occurred along with increased sulfide production. The presence of 1 mM cysteine and (or) 1 mM cysteine and (or) 1 mM methionine does not affect the inverse sulfur isotope effect for evolved H2S. However, 5 and 10 mM cysteine reduce the maximum delta34S value for released H2S from +40 to 10%. A small conversion of cysteine to H2S by C. pasteurianum occurs, but only in the stationary phase.     

Isolation and Properties of Selenomethionine-Resistant Mutants of NEUROSPORA CRASSA

Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-1(r) locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-1(r). They are resistant not only to selenomethionine but also to ethionine, while the ethionine-resistant mutant, eth-1(r), is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.-It is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-met(r) mutants is not repressed by a high concentration of methionine (5 mM), although inorganic sulfate (2 mM) still can cause total repression. The gamma-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that of the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase.     

ATP sulfurylase from trophosome tissue of Riftia pachyptila (hydrothermal vent tube worm)

ATP sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) was extensively purified from trophosome tissue of Riftia pachyptila, a tube worm that thrives in deep ocean hydrothermal vent communities. The enzyme is probably derived from the sulfide-oxidizing bacteria that densely colonize the tissue. Glycerol (20% v/v) protected the enzyme against inactivation during purification and storage. The native enzyme appears to be a dimer (MW 90 kDa +/- 10%) composed of identical size subunits (MW 48 kDa +/- 5%). At pH 8.0, 30 degrees C, the specific activities (units x mg protein-1) of the most highly purified sample are as follows: ATP synthesis, 370; APS synthesis, 23; molybdolysis, 65; APSe synthesis or selenolysis, 1.9. The Km values for APS and PPi at 5 mM Mg2+ are 6.3 and 14 microM, respectively. In the APS synthesis direction, the Km values for MgATP and SO4(2-) are 1.7 and 27 mM, respectively. The Km values for MgATP and MoO4(2-) in the molybdolysis reaction are 80 and 150 microM, respectively. The Kia for MgATP is 0.65 mM. APS is a potent inhibitor of molybdolysis, competitive with both MgATP and MoO4(2-) (Kiq = 2.2 microM). However, PPi (+ Mg2+) is virtually inactive as a molybdolysis inhibitor. Oxyanion dead end inhibitors competitive with SO4(2-) include (in order of decreasing potency) ClO4- greater than FSO3- (Ki = 22 microM) greater than ClO3- greater than NO3- greater than S2O3(2-) (Ki's = 5 and 43 mM). FSO3- is uncompetitive with MgATP, but S2O3(2-) is noncompetitive. Each subunit contains two free SH groups, at least one of which is functionally essential. ATP, MgATP, SO4(2-), MoO4(2-), and APS each protect against inactivation by excess 5,5'-dithiobis-(2-nitrobenzoate). FSO3- is ineffective as a protector unless MgATP is present. PPi (+Mg2+) does not protect against inactivation. Riftia trophosome contains little or no "ADP sulfurylase." The high trophosome level of ATP sulfurylase (67-176 ATP synthesis units x g fresh wt tissue-1 from four different specimens, corresponding to 4-10 microM enzyme sites), the high kcat of the enzyme for ATP synthesis (296 s-1), and the high Km's for MgATP and SO4(2-) are consistent with a role in ATP formation during sulfide oxidation, i.e., the physiological reaction is APS + MgPPi in equilibrium SO4(2-) + MgATP.     

Biosynthesis of biopterin. Studies on the mechanism of 6-pyruvoyltetrahydropteridine synthase

[1'-3H]- and [2'-3H]dihydroneopterin triphosphate (NH2TP) were prepared enzymatically from [4-3H]- and [5-3H]glucose and converted to tetrahydrobiopterin (BH4) by an extract from bovine adrenal medulla. The formation of BH4 from both [1'-3H]- and [2'-3H]-NH2TP proceeds with virtually complete loss of the respective tritium label. The breaking of the CH-bond at C-1' is characterized by a kinetic isotope effect of 2.6 +/- 0.5. A smaller kinetic isotope effect of 1.5 +/- 0.2 was found for the breaking of the CH-bond at C-2'.     

Se-(8-azidoadenosyl)[75Se]selenomethionine as a photoaffinity label for S-adenosylmethionine binding proteins.

A method is described for the synthesis and purification of the photoaffinity label Se-(8-azidoadenosyl)[75Se]selenomethionine. This photoaffinity label can be used to specifically and covalently label the S-adenosylmethionine binding site of proteins that use this cofactor, as exemplified by labeling of thioether methyltransferase. By utilizing the gamma-emitting isotope of selenium, Se-(8-azidoadenosyl)[75Se]selenomethionine eliminates the need for the impregnation of acrylamide gels with fluorographic enhancers and dilution of liquid samples into scintillation cocktails, as is required with the commonly used methyl-3H-labeled and 35S-labeled S-(8-azidoadenosyl)methionine.     

Isotope partitioning in the adenosine 3',5'-monophosphate dependent protein kinase reaction indicates a steady-state random kinetic mechanism

Isotope partitioning beginning with the binary E.MgATP and E.N-acetyl-Leu-Arg-Arg-Ala-Ser-Leu-Gly (Ser-peptide) complexes indicates that the kinetic mechanism for the adenosine 3',5'-monophosphate dependent protein kinase is steady-state random. A total of 100% of the initial radioactive E.MgATP complex is trapped as phospho-Ser-peptide at infinite Ser-peptide concentration at both low and high concentration of uncomplexed Mg2+, suggesting that the off-rate of MgATP from the E.MgATP.Ser-peptide complex is slow relative to the catalytic steps. Km for Ser-peptide in the trapping reaction decreases from 17 microM at low Mg2+ to 2 microM at high Mg2+, indicating that Mg2+ decreases the off-rate for MgATP from the E.MgATP complex. A total of 100% of the radioactive E.Ser-peptide complex is trapped as phospho-Ser-peptide at low Mg2+, but only 40% is trapped at high Mg2+ in the presence of an infinite concentration of MgATP, suggesting that the off-rate for Ser-peptide from the central complex is much less than catalysis at low but not at high Mg2+. In support of this finding, the Ki for Leu-Arg-Arg-Ala-Ala-Leu-Gly (Ala-peptide) increases from 0.27 mM at low Mg2+ to 2.4 mM at high Mg2+. No trapping was observed at either high or low Mg2+ for the E.MgADP complex up to a phospho-Ser-peptide concentration of 5 mM. Thus, it is likely that in the slow-reaction direction the kinetic mechanism is rapid equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discrimination of assembled and disassembled forms of gizzard myosin by papain

Chicken gizzard myosin in 0.15 M or 0.5 M NaCl was cleaved at two sites of heavy chain with 2-10 micrograms/ml papain. MgATP inhibited these cleavages of myosin in 0.15 M NaCl but not in 0.5 M NaCl. The protective effect of ATP was observed at concentrations as low as 10 microM and increased in proportion to ATP concentration to a maximum at 1 mM. ADP was as effective as ATP, while adenosine 5'-[beta, gamma-imido]triphosphate, an unhydrolyzable ATP analogue, was less effective than ATP or ADP. AMP had no protective effect on the digestion of myosin and GTP inhibited slightly the digestion. When the papain-insensitive myosin in 0.15 M NaCl and 2.5 mM MgATP was phosphorylated by Ca2+/calmodulin-dependent myosin light-chain kinase, the myosin restored the vulnerability to papain. However, the two papain-susceptible forms, nonphosphorylated form in the absence of MgATP and phosphorylated form in the presence of MgATP, yielded very similar but distinct proteolytic fragments upon the digestion. When the extent of myosin assembly was estimated by the turbidimetry of myosin suspension in 0.15 M NaCl, nonphosphorylated myosin in the absence and presence of MgATP was assembled and disassembled, respectively, and phosphorylated myosin in the presence of MgATP was assembled. These results suggest that, at physiological ionic strength, papain as a probe distinguishes disassembled myosin and assembled myosin as papain-insensitive and papain-sensitive forms, respectively.     

Calcium release from two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium release from sarcoplasmic reticulum vesicles presumably derived from longitudinal tubules (LSR) and terminal cisternae (HSR) of rabbit skeletal muscle was investigated by dual wavelength spectrophotometry using the calcium-indicator antipyrylazo III. In 120 mM KCl, 5 mM MgCl2, 30 microM, CaCl2, 50 microM MgATP, 100 microM antipyrylazo III, 40 mM histidine (pH 6.8, 25 degrees C), LSR and HSR sequestered approx. 115 nmol calcium/mg, and then spontaneously released calcium. Analysis of ATP hydrolysis and phosphoenzyme level during LSR and HSR calcium sequestration indicated that this calcium release process was passive, occurring in the virtual absence of ATP and phosphoenzyme. Moreover, subsequent addition of ATP reinitiated the calcium sequestration-release sequence. Calcium release by HSR was more than 4-times faster than that by LSR. Analysis of the calcium release phase demonstrated a biexponential decay for both LSR (0.10 and 0.63 min-1) and HSR (0.26 and 1.65 min-1), suggestive of heterogeneity within each fraction. Replacement of 120 mM KCl with either 120 mM choline chloride, 240 mM sucrose, or H2O reduced maximal calcium sequestration by LSR, but had less effect on LSR calcium release rate constants. In the case of HSR, these changes in the ionic composition of the medium drastically reduced calcium release rate constants with little effect on calcium content. These marked differences between LSR and HSR are consistent with the hypothesis that the calcium permeability of the terminal cisternae is greater and more sensitive to the ionic environment than is that of the longitudinal tubules of sarcoplasmic reticulum.