Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Determining ligand dissociation constants of binary and ternary complexes from the kinetics of enzyme inactivation

Adenosine 5-phosphosulfate (APS) kinase from Penicillium chrysogenum is irreversibly inactivated by trinitrobenzene sulfonate in a pseudo-first order process. Under standard assay conditions kapp was 1.9 X 10(-3) s-1. Saturating MgATP or MgADP decreased Kapp to a limit of 4.1 X 10(-4) s-1. There are several explanations for the partial protection, including the presence of two essential lysyl side chains, only one of which is at the active site. Analysis of the inactivation kinetics by means of linear plots derived for partial protection yielded dissociation constants for E X MgATP (Kia) and E X MgADP (Kiq) of 2.9 mM and 1.8 mM, respectively. Low concentrations of APS alone provided no protection against trinitrobenzene sulfonate inactivation, but in the presence of 1 mM MgADP, as little as 2 microM APS provided additional protection while 100 microM APS reduced kapp to the limit of 4.1 X 10(-4) s-1. The results confirm the formation of a dead end E X MgADP X APS proposed earlier as the cause of the potent substrate inhibition by APS. Linear plots of 1/delta k versus 1/[MgADP] at different fixed [APS] and of 1/delta k versus 1/[APS] at different fixed [MgADP] were characteristic of the ordered binding of MgADP before APS (or the highly synergistic random binding of the two ligands). The true APS dissociation constant of the dead end E X MgADP X APS complex (K'ib) was determined to be 1.9 microM. From the value of K'ib and the previously reported value of KIB (apparent inhibition constant of APS as a substrate inhibitor of the catalytic reaction at saturating MgATP), the ratio of the MgADP and PAPS release rate constants (k4/k3) was calculated to be 11. Inactivation kinetics was used to study the effects of Mg2+ and high salt on ADP and APS binding. The results indicated that free ADP binds to the enzyme more tightly than does MgADP at low ionic strength. High salt decreased free ADP binding, but had little effect on MgADP binding. APS binds more tightly to E X MgADP in the absence or presence of salt than to E X ADP.     

Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Purification and kinetic characterization

Adenosine 5'-phosphosulfate (APS) kinase, the second enzyme in the pathway of inorganic sulfate assimilation, was purified to near homogeneity from mycelium of the filamentous fungus, Penicillium chrysogenum. The enzyme has a native molecular weight of 59,000-60,000 and is composed of two 30,000-dalton subunits. At 30 degrees C, pH 8.0 (0.1 M Tris-chloride buffer), 5.5 microM APS, 5 mM MgATP, 5 mM excess MgCl2, and "high" salt (70-150 mM (NH4)2SO4), the most highly purified preparation has a specific activity of 24.7 units X mg of protein-1 in the physiological direction of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) formation. This activity is nearly 100-fold higher than that of any previously purified preparation of APS kinase. APS kinase is subject to potent substrate inhibition by APS. In the absence of added salt, the initial velocity at 5 mM MgATP plus 5 mM Mg2+ is maximal at about 1 microM APS and half-maximal at 0.2 and 4.4 microM APS. In the presence of 200 mM NaCl or 70-150 mM (NH4)2SO4, the optimum APS concentration shifts to 4-6 microM APS; the half-maximal values shift to 1-1.3 and 21-27 microM APS. The steady state kinetics of the reaction were investigated using a continuous spectrophotometric assay. The families of reciprocal plots in the range 0.25-5 mM MgATP and 0.8-5.1 microM APS are linear and intersect on the horizontal axis. Appropriate replots yield KmMgATP = 1.5 mM, KmAPS = 1.4 microM, and Vmax, = 38.7 units X mg of protein-1. Excess APS is an uncompetitive inhibitor with respect to MgATP (K1APS = 23 microM). PAPS, the product of the forward reaction, is also uncompetitive with MgATP. PAPS is not competitive with APS. In the reverse direction, the plots have the characteristics of a rapid equilibrium ordered sequence with MgADP adding before PAPS. The kinetic constants are KmPAPS = 8 microM, KiMgADP = 560 microM, and Vmaxr = 0.16 units X mg of protein-1. Iso-PAPS (the 2'-phosphate isomer of PAPS) is competitive with PAPS and uncompetitive with respect to MgADP (Ki = 6 microM). APS kinase is inactivated by phenylglyoxal, suggesting the involvement of an essential argininyl residue. MgATP or MgADP at 10 Ki protect against inactivation. APS or PAPS at 600 and 80 Km, respectively, are ineffective alone, but provide nearly complete protection in the presence of 0.1 Ki of MgADP or MgATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenosine-5'-phosphosulfate kinase from Escherichia coli K12. Purification, characterization, and identification of a phosphorylated enzyme intermediate

Adenosine-5'-phosphosulfate kinase (ATP:adenylylsulfate 3'-phosphotransferase), the second enzyme in the pathway of sulfate activation, has been purified (approximately 300-fold) to homogeneity from an Escherichia coli K12 strain, which overproduces the enzyme activity (approximately 100-fold). The purified enzyme has a specific activity of 153 mumol of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) formed/min/mg of protein at 25 degrees C. The enzyme is remarkably efficient with a Vmax/Km(APS) of greater than 10(8) M-1 s-1, indicating that at physiologically low substrate concentrations the reaction is essentially diffusion limited. Upon incubation with MgATP a phosphorylated enzyme is formed; the isolated phosphorylated enzyme can transfer its phosphoryl group to adenosine 5'-phosphosulfate (APS) to form PAPS or to ADP to form ATP. The phosphorylated enzyme exists as a dimer of identical 21-kilodalton subunits, while the dephosphorylated form primarily exists as a tetramer. Divalent cations are required for activity with Mg(II), Mn(II), Co(II), and Cd(II) activating. Studies of the divalent metal-dependent stereoselectivity for the alpha- and beta-phosphorothioate derivatives of ATP indicate metal coordination to at least the alpha-phosphoryl group of the nucleotide. Steady state kinetic studies of the reverse reaction indicate a sequential mechanism, with a rapid equilibrium ordered binding of MgADP before PAPS. In the forward direction APS is a potent substrate inhibitor, competitive with ATP, complicating kinetic studies. The primary kinetic mechanism in the forward direction is sequential. Product inhibition studies at high concentrations of APS suggest an ordered kinetic mechanism with MgATP binding before APS. At submicromolar concentrations of APS, product inhibition by both MgADP and PAPS is more complex and is not consistent with a solely ordered sequential mechanism. The formation of a phosphorylated enzyme capable of transferring its phosphoryl group to APS or to MgADP suggests that a ping-pong pathway in which the rate of MgADP dissociation is comparable to the rate of APS binding might contribute at very low concentrations of APS. The substrate inhibition by APS is consistent with APS binding to the enzyme, to form a dead-end E.APS complex.     

Human 3'-phosphoadenosine 5'-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase domains

Recombinant human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase, isoform 1 (brain), was purified to near-homogeneity from an Escherichia coli expression system and kinetically characterized. The native enzyme, a dimer with each 71 kDa subunit containing an adenosine triphosphate (ATP) sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain, catalyzes the overall formation of PAPS from ATP and inorganic sulfate. The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol. APS kinase activity displayed the characteristic substrate inhibition by APS (K(I) of 47.9 microM at saturating MgATP). The maximum attainable activity of 0.12 micromol min(-1) (mg of protein)(-1) was observed at an APS concentration ([APS](opt)) of 15 microM. The theoretical K(m) for APS (at saturating MgATP) and the K(m) for MgATP (at [APS](opt)) were 4.2 microM and 0.14 mM, respectively. At likely cellular levels of MgATP (2.5 mM) and sulfate (0.4 mM), the overall endogenous rate of PAPS formation under optimum assay conditions was 0.09 micromol min(-1) (mg of protein)(-1). Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 micromol min(-1) (mg of protein)(-1). The kinetic constants of the ATP sulfurylase domain were as follows: V(max,f) = 0.77 micromol min(-1) (mg of protein)(-1), K(mA(MgATP)) = 0.15 mM, K(ia(MgATP)) = 1 mM, K(mB(sulfate)) = 0.16 mM, V(max,r) = 18.7 micromol min(-1) (mg of protein)(-1), K(mQ(APS)) = 4.8 microM, K(iq(APS)) = 18 nM, and K(mP(PPi)) = 34.6 microM. The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) accumulation of APS in solution during the overall reaction, (c) rate acceleration provided by exogenous APS kinase, and (d) availability of both active sites to exogenous APS all argue against APS channeling. Molybdate, selenate, chromate ("chromium VI"), arsenate, tungstate, chlorate, and perchlorate bind to the ATP sulfurylase domain, with the first five serving as alternative substrates that promote the decomposition of ATP to AMP and PP(i). Selenate, chromate, and arsenate produce transient APX intermediates that are sufficiently long-lived to be captured and 3'-phosphorylated by APS kinase. (The putative PAPX products decompose to adenosine 3',5'-diphosphate and the original oxyanion.) Chlorate and perchlorate form dead-end E.MgATP.oxyanion complexes. Phenylalanine, reported to be an inhibitor of brain ATP sulfurylase, was without effect on PAPS synthetase isoform 1.     

Direct photoaffinity labeling of proteins with adenosine 3'-[32P]phosphate 5'-phosphosulfate. Atractyloside inhibits labeling of a Mr = 34,000 protein in an adrenal medullary Golgi fraction

Direct photoaffinity labeling with radioactively labeled adenosine 3'-phosphate 5'-phosphosulfate (PAPS) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography was used to identify PAPS binding proteins in a Golgi membrane preparation of bovine adrenal medulla. [3'-32P]PAPS was synthesized from adenosine 5'-phosphosulfate (APS) and [gamma-32P]ATP using APS kinase prepared from yeast and was purified by reverse-phase ion pair high performance liquid chromatography. Upon irradiation with UV light, [3'-32P]PAPS, as well as [35S]PAPS under conditions which minimized sulfotransferase-catalyzed incorporation of 35SO4 from [35S]PAPS into proteins, bound selectively to a 34-kDa protein of the Golgi membrane preparation. PAPS binding to the 34-kDa protein was strongly inhibited by the presence of 50 microM atractyloside. The 34-kDa PAPS binding protein therefore appears to be similar to the mitochondrial ATP/ADP translocator with regard to both molecular weight and inhibition by atractyloside of adenine nucleotide binding. Photoaffinity labeling will be useful in the purification and functional identification of the 34-kDa protein.     

Properties and Localization of the Sulfate-Activating System in Rat Brain

The formation of the sulfate donor [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) from inorganic [35S]sulfate was studied using a novel assay. The assay was based on the quantitative transfer of radioactivity from [35S]PAPS to beta-naphthol under the action of phenolsulfotransferase activity from rat brain cytosol, with the [35S]beta-naphthyl sulfate formed being isolated by polystyrene bead chromatography. This simple assay was validated by comparison of results with those derived from direct assay of [35S]PAPS isolated by either TLC or ion exchange chromatography. [35S]PAPS formation by a high-speed supernatant of rat cerebral cortex occurred with an optimal pH of approximately 7.6, varied linearly with time and protein concentration, and depended on the presence of Mg2+-ATP. The latter could not be replaced by other nucleotides such as GTP, UTP, or CTP, which at 1-5 mM concentrations inhibited the reaction. Mg2+ could not be replaced by Mn2+, which at micromolar concentrations inhibited the reaction. The apparent Km values of Mg2+-ATP (at 0.1 mM [35S]sulfate) and inorganic sulfate (at 5 mM Mg2+-ATP) were 2.7 and 0.2 mM, respectively. These kinetics parameters corresponded to those reported for purified ATP sulfurylase (EC 2.7.7.4), the enzyme responsible for the first step of PAPS synthesis in liver. The product of its reaction, [35S]adenosine 5'-phosphosulfate (APS), could not be detected after incubations, an observation implying that the action of APS kinase was not rate limiting in cerebral extracts tested under the selected experimental conditions. [35S]PAPS formation was detectable in cytosolic fractions from various brain regions, which displayed only limited differences in synthesizing activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the affinity of each component of a composite quaternary transition-state analogue complex of creatine kinase

Recombinant rabbit muscle creatine kinase (CK) was titrated with MgADP in 50 mM Bicine and 5 mM Mg(OAc)2, pH 8.3, at 30.0 degrees C by following a decrease in the protein's intrinsic fluorescence. In the presence of 50 mM NaOAc, but in the absence of added creatine or nitrate, MgADP has an apparent K(d) of 135 +/- 7 microM, and the total change in fluorescence on saturation (Delta%F) is 15.3 +/- 0.3%. Acetate was used as the anion in this experiment because it does not promote the formation of a CK.MgADP.anion.creatine transition-state analogue complex (TSAC) [Millner-White and Watts (1971) Biochem. J. 122, 727-740]. In the presence of 80 mM creatine, but no nitrate, the apparent K(d) for MgADP remains essentially unchanged at 132 +/- 10 microM, while Delta%F decreases slightly to 13.2 +/- 0.3%. In the presence of 10 mM nitrate, but no creatine, the apparent K(d) is once again essentially unchanged at 143 +/- 23 microM, but the Delta%F is markedly reduced to 4.2 +/- 0.2%. The presence of both 10 mM nitrate and 80 mM creatine during titration reduces the apparent K(d) for MgADP 10-fold to 13.7 +/- 0.7 microM, and Delta%F increases to 20.6 +/- 0.3%, strongly suggesting that the simultaneous presence of saturating levels of creatine and nitrate increases the affinity of CK for MgADP and promotes the formation of the enzyme*MgADP*nitrate*creatine TSAC. When the fluorescence of CK was titrated with MgADP in the presence of 80 mM creatine and fixed saturating concentrations of various anions, apparent K(d) values for MgADP of 132 +/- 10 microM, 25.2 +/- 1.3 microM, 18.8 +/- 0.9 microM, 13.7 +/- 0.7 microM, and 6.4 +/- 0.7 microM were observed as the anion was changed from acetate to formate to chloride to nitrate to nitrite, respectively. This is the same trend reported by Millner-White and Watts for the effectiveness of various monovalent anions in forming the CK.MgADP.anion.creatine TSAC. On titration of CK with MgADP in the presence of 80 mM creatine and various fixed concentrations of NaNO3, the apparent K(d) for MgADP decreases with increasing fixed concentrations of nitrate. A plot of the apparent K(d) for MgADP vs [NO3-] suggests a K(d) for nitrate from the TSAC of 0.39 +/- 0.07 mM. Similarly, titration with MgADP in the presence of 10 mM NaNO3 and various fixed concentrations of creatine gives a value of 0.9 +/- 0.4 mM for the dissociation of creatine from the TSAC. The data were used to calculate K(TDAC), the dissociation constant of the quaternary TSAC into its individual components, of 3 x 10(-10) M3. To our knowledge this is the first reported dissociation constant for a ternary or quaternary TSAC.     

Purification and characterization of bile salt sulfotransferase from human liver

Bile salt sulfotransferase, the enzyme responsible for the formation of bile salt sulfate esters, was purified extensively from normal human liver. The purification procedure included DEAE-Sephadex chromatography, taurocholate-agarose affinity chromatography, and preparative isoelectrofocusing. The final preparation had a specific activity of 18 nmol min-1 mg protein-1, representing a 760-fold purification from the cytosol fraction with a overall yield of 15%. The human enzyme has a Mr of 67,000 and a pI of 5.2. DEAE-Sephadex chromatography of the cytosol fraction revealed only a single species of activity. The limiting Km for the sulfuryl donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), is 0.7 microM. The limiting Km for the sulfuryl acceptor, glycolithocholate (GLC), is 2 microM. Reciprocal plots were intersecting. Product inhibition studies established that adenosine 3',5'-diphosphate (PAP) was competitive with PAPS (Ki = 0.2 microM) and noncompetitive with respect to GLC. GLC sulfate was competitive with GLC (Ki = 2.2 microM) and noncompetitive with respect to PAPS. Also, 3-ketolithocholate, a dead-end inhibitor, was competitive with GLC (Ki = 0.6 microM) and noncompetitive with respect to PAPS. Iso-PAP (the 2' isomer of PAP) was competitive with PAPS (Ki = 0.3 microM) and noncompetitive with GLC. The cumulative results of the steady-state kinetics experiments point to a random mechanism for the binding of substrates and release of products. The purified enzyme displays no activity toward estrone, testosterone, or phenol. Among the reactive substrates tested, the Vmax/Km values are in the order GLC greater than 3-beta OH-5-cholenic acid greater than glycochenodeoxycholate greater than glycocholate. p-Chloromercuribenzoate inactivated the enzyme. Either PAPS or GLC protected against inactivation, suggesting the presence of a sulfhydryl group at the active site.     

Spectrofluorimetric analysis of 7-hydroxycoumarin binding to bovine phenol sulfotransferase

Phenol sulfotransferases (SULT1s, EC 2.8.2.1) catalyze sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl oxygen of aromatic acceptor substrates. Previous work with the bovine SULT1A1 has utilized the highly fluorescent substrate 7-hydroxycoumarin (7-HC, umbelliferone) as an acceptor substrate [Biochem. Biophys. Res. Commun. 261 (1999) 815]. Here we report that adenosine-3',5'-bisphosphate (PAP)-dependent binding of 7-HC to bSULT1A1 can be observed due to the appearance of a 400-420-nm shoulder in the emission spectrum, using an excitation wavelength of 280 nm. This emission was observed by placing 7-HC in ethanol, which is consistent with bSULT1A1 phenol binding site hydrophobicity. Titrations with 7-HC indicate a K(d) for 7-HC of 0.58 microM and substoichiometric binding to the homodimeric enzyme. The bSULT1A1:PAP:7-HC complex could be disrupted with pentachlorophenol (PCP), titrations with which indicated 0.5 equivalents per enzyme subunit. Titrations of enzyme plus 7-HC with PAP also indicated 0.5 equivalents per enzyme subunit. These results suggest a model of homodimeric bSULT1A1 in which subunit interactions favor half-site reactivity in the formation of a dead end complex.     

Reduction of adenosine-5'-phosphosulfate instead of 3'-phosphoadenosine-5'-phosphosulfate in cysteine biosynthesis by Rhizobium meliloti and other members of the family Rhizobiaceae.

We have cloned and sequenced three genes from Rhizobium meliloti (Sinorhizobium meliloti) that are involved in sulfate activation for cysteine biosynthesis. Two of the genes display homology to the Escherichia coli cysDN genes, which code for an ATP sulfurylase (EC 2.7.7.4). The third gene has homology to the E. coli cysH gene, a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase (EC 1.8.99.4), but has greater homology to a set of genes found in Arabidopsis thaliana that encode an adenosine-5'-phosphosulfate (APS) reductase. In order to determine the specificity of the R. meliloti reductase, the R. meliloti cysH homolog was histidine tagged and purified, and its specificity was assayed in vitro. Like the A. thaliana reductases, the histidine-tagged R. meliloti cysH gene product appears to favor APS over PAPS as a substrate, with a Km for APS of 3 to 4 microM but a Km for PAPS of >100 microM. In order to determine whether this preference for APS is unique to R. meliloti among members of the family Rhizobiaceae or is more widespread, cell extracts from R. leguminosarum, Rhizobium sp. strain NGR234, Rhizobium fredii (Sinorhizobium fredii), and Agrobacterium tumefaciens were assayed for APS or PAPS reductase activity. Cell extracts from all four species also preferentially reduce APS over PAPS.     

Translocation of adenosine 3'-phosphate 5'-phosphosulfate into rat liver Golgi vesicles

The translocation of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) across rat liver Golgi-derived vesicles has been studied. Vesicles of the same topographical orientation as in vivo were incubated with a mixture of [adenine-8-3H]PAPS and [35S]PAPS. The tritium to radiolabeled sulfur ratio of the incubation medium was 1.73 +/- 0.03 while that in the vesicles was 1.82 +/- 0.13. This strongly suggests that the entire PAPS molecule was being translocated across the Golgi vesicle membrane even though intact PAPS could not be detected within the vesicles. Translocation of PAPS resulted in accumulation of solutes within vesicles. This accumulation was temperature dependent, saturable (apparent Km = 0.7 microM; Vmax = 25 pmol/mg of protein/10 min), and inhibited by the substrate analogue 3',5'-ADP but not by 2',5'-ADP. Translocation of PAPS was inhibited following treatment of Golgi vesicles with Pronase under conditions in which the activity of a lumenal Golgi membrane marker such as sialyltransferase was not. This result is consistent with the existence of a PAPS carrier protein, portions of which face the cytoplasmic side of the Golgi membrane.     

Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants : use of adenosine-5'-phosphosulfate and adenosine-3'-phosphate 5'-phosphosulfate as precursors

Adenosine-5′-phosphosulfate (APS) and adenosine-3′-phosphate 5′-phosphosulfate (PAPS) have been used as precursors of sulfoquinovosyldiacylglycerol (SQDG) in intact chloroplasts incubated in the dark. Competition studies demonstrated APS was preferred over PAPS and SO₄²⁻. Rates of SQDG synthesis up to 3 nanomoles per milligram of chlorophyll per hour were observed when [³⁵S]APS and appropriate cofactors were supplied to chloroplasts incubated in the dark. The pH optimum for utilization of APS was 7.0. The incorporation was linear for at least 30 minutes. ATP and UTP stimulated the incorporation of sulfur from APS into SQDG, but the most stimulatory additions were DHAP and glycerol-3-P. The concentration curve for APS showed a maximum at 20 micromolar in the absence of DHAP and 30 micromolar in the presence of DHAP. The optimum concentration of DHAP for conversion of APS into SQDG was 2 millimolar. Rates of synthesis up to 4 nanomoles per milligram of chlorophyll per hour were observed when [³⁵S]PAPS was the sulfur donor and appropriate cofactors were supplied to chloroplasts. Optimal rates for conversion of sulfur from PAPS into SQDG occurred with concentrations of DHAP between 5 and 10 millimolar. DHAP was by far the most effective cofactor, although ATP and UTP also stimulated the utilization of PAPS for SQDG biosynthesis. In general, triose phosphates, including glycerol-3-P were not effective cofactors for SQDG biosynthesis.     

Adenine nucleotides affect the binding of 3-phosphoglycerate to pig muscle 3-phosphoglycerate kinase

Pig muscle 3-phosphoglycerate kinase was complexed with 1-anilino-8-naphthalenesulfonate (ANS) in order to monitor the binding of substrates to the enzyme. The enzyme-dye interaction did not influence the enzymic activity under the experimental conditions used. By measuring the substrate-dependent change in the fluorescence emission of ANS molecules tightly bound to the enzyme (Kd less than or equal to 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20 degrees C. The dissociation constants obtained for the separate bindings of 3-phosphoglycerate, MgATP, 1,3-bisphosphoglycerate and MgADP were 0.03 +/- 0.01 mM, 0.15 +/- 0.10 mM, 0.00005 +/- 0.00001 mM and 0.15 +/- 0.10 mM respectively. binding of 3-phosphoglycerate is weakened when MgATP is also bound to the enzyme: the dissociation constant of 3-phosphoglycerate in this ternary complex (0.25 +/- 0.08 mM) is comparable to its Km value (0.38 +/- 0.10 mM). The same weakening can be observed in the non-productive ternary complexes where MgATP is replaced by MgADP (Kd = 0.20 +/- 0.10 mM) or AMP (Kd = 0.12 +/- 0.05 mM), whereas adenosine has no such effect. This indicates the importance of the negatively charged phosphate(s) of nucleotides in influencing the binding of 3-phosphoglycerate. In contrast to 3-phosphoglycerate, the binding of the substrate analogue, glycerol 3-phosphate is practically not affected by the presence of MgATP: the dissociation constant to the free enzyme (0.40 +/- 0.10 mM) is comparable to its inhibitory constant (0.70 +/- 0.20 mM). This finding and the similarity of the dissociation constant of glycerol 3-phosphate binding (0.40 +/- 0.10 mM) and the Km value of 3-phosphoglycerate (0.38 +/- 0.10 mM) suggest that, during the enzymic reaction, binding of 3-phosphoglycerate occurs probably without involvement of the carboxyl group.     

APS kinase from Penicillium chrysogenum. Dissociation and reassociation of subunits as the basis of the reversible heat inactivation

Adenosine-5'-phosphosulfate (APS) kinase from Penicillium chrysogenum, loses catalytic activity at temperatures greater than approximately 40 degrees C. When the heat-inactivated enzyme is cooled to 30 degrees C or lower, activity is regained in a time-dependent process. At an intermediary temperature (e.g. 36 degrees C) an equilibrium between active and inactive forms can be demonstrated. APS kinase from P. chrysogenum is a dimer (Mr = 57,000-60,000) composed of two apparently identical subunits. Three lines of evidence suggest that the reversible inactivation is a result of subunit dissociation and reassociation. (a) Inactivation is a first-order process. The half-time for inactivation at a given temperature is independent of the original enzyme concentration. Reactivation follows second-order kinetics. The half-time for reactivation is inversely proportional to the original enzyme concentration. (b) The equilibrium active/inactive ratio at 36 degrees C increases as the total initial enzyme concentration is increased. However, Keq,app at 5 mM MgATP and 36 degrees C calculated as [inactive sites]2/0.5 [active sites] is near-constant at about 1.7 X 10(-8) M over a 10-fold concentration range of enzyme. (c) At 46 degrees C, the inactive P. chrysogenum enzyme (assayed after reactivation) elutes from a calibrated gel filtration column at a position corresponding to Mr = 33,000. Substrates and products of the APS kinase reaction had no detectable effect on the rate of inactivation. However, MgATP and MgADP markedly stimulated the reactivation process (kapp = 3 X 10(5) M-1 X s-1 at 30 degrees C and 10 mM MgATP). The kapp for reactivation was a nearly linear function of MgATP up to about 20 mM suggesting that the monomer has a very low affinity for the nucleotide compared to that of the native dimer. Keq,app at 36 degrees C increases as the MgATP concentration is increased. The inactivation rate constant increased as the pH was decreased but no pK alpha could be determined. The reactivation rate constant increased as the pH was increased. An apparent pK alpha of 6.4 was estimated.     

Crystal structure of adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum

Adenosine 5'-phosphosulfate (APS) kinase catalyzes the second reaction in the two-step conversion of inorganic sulfate to 3'-phosphoadenosine 5'-phosphosulfate (PAPS). This report presents the 2.0 A resolution crystal structure of ligand-free APS kinase from the filamentous fungus, Penicillium chrysogenum. The enzyme crystallized as a homodimer with each subunit folded into a classic kinase motif consisting of a twisted, parallel beta-sheet sandwiched between two alpha-helical bundles. The Walker A motif, (32)GLSASGKS(39), formed the predicted P-loop structure. Superposition of the APS kinase active site region onto several other P-loop-containing proteins revealed that the conserved aspartate residue that usually interacts with the Mg(2+) coordination sphere of MgATP is absent in APS kinase. However, upon MgATP binding, a different aspartate, Asp 61, could shift and bind to the Mg(2+). The sequence (156)KAREGVIKEFT(166), which has been suggested to be a (P)APS motif, is located in a highly protease-susceptible loop that is disordered in both subunits of the free enzyme. MgATP or MgADP protects against proteolysis; APS alone has no effect but augments the protection provided by MgADP. The results suggest that the loop lacks a fixed structure until MgATP or MgADP is bound. The subsequent conformational change together with the potential change promoted by the interaction of MgATP with Asp 61 may define the APS binding site. This model is consistent with the obligatory ordered substrate binding sequence (MgATP or MgADP before APS) as established from steady state kinetics and equilibrium binding studies.     

Spectroscopic studies of the interactions of coenzymes and coenzyme fragments with pig heart, oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase

Spectroscopic, ultrafiltration, and kinetic studies have been used to characterize interactions of reduced and oxidized triphosphopyridine nucleotides (TPNH and TPN), 2'-phosphoadenosine 5'-diphosphoribose (Rib-P2-Ado-P), and adenosine 2',5'-bisphosphate [Ado(2',5')P2] with with TPN-specific isocitrate dehydrogenase. Close similarity of the UV difference spectra and of the protein fluorescence changes accompanying the formation of the binary complexes provides evidence for the binding of these nucleotides to the same site on the enzyme. From the pH dependence of the dissociation constants for TPNH binding to TPN-specific isocitrate dehydrogenase in the absence and in the presence of Mn2+, over the pH range 5.8-7.6, it has been demonstrated that the nucleotide binds to the enzyme in its unprotonated, metal-free form. The involvement of positively charged residues, protonated over the pH range studied, has been postulated. One TPNH binding site per enzyme subunit has been measured by fluorescence and difference absorption titrations. A dramatic effect of ionic strength on binding has been demonstrated: about a 1000-fold decrease in the dissociation constant for TPNH has been observed at pH 7.6 upon decreasing ionic strength from 0.336 (Kd = 1.2 +/- 0.2 microM) to 0.036 M (Kd = 0.4 +/- 0.1 nM) in the presence and in the absence of 100 mM Na2SO4, respectively. Weak competition of sulfate ions for the nucleotide binding site has been observed (KI = 57 +/- 3 mM). The binding of TPN in the presence of 100 mM Na2SO4 at pH 7.6 is about 100-fold weaker (Kd = 110 +/- 22 microM) than the binding of the reduced coenzyme and is similarly affected by ionic strength. These results demonstrate the importance of electrostatic interactions in the binding of the coenzyme to TPN-specific isocitrate dehydrogenase. The large enhancement of protein fluorescence caused by binding of TPN and Rib-P2-Ado-P (delta Fmax = 50%) and of Ado(2',5')P2 (delta Fmax = 41%) has been ascribed to a local conformational change of the enzyme. An apparent stoichiometry of 0.5 nucleotide binding site per peptide chain was determined for TPN, Rib-P2-Ado-P, and Ado(2',5')P2 from fluorescence titrations, in contrast to one binding site per enzyme subunit determined from UV difference spectral titration and ultrafiltration experiments. Thus, the binding of one molecule of the nucleotide per dimeric enzyme molecule is responsible for the total increase in protein fluorescence, while binding to the second subunit does not cause further change.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cholecystokinin activation: evidence for an ordered reaction mechanism for the tyrosyl protein sulfotransferase responsible for the peptide sulfation.

The kinetics of the forward tyrosyl protein sulfotransferase (TPS) reaction were examined using an assay based on the 35SO4 transfer from 3'-phosphoadenosine 5'-phospho(35S)sulfate [( 35S]PAPS) to tyrosyl residues of the non-sulfated cholecystokinin derivative, BocCCK-8(ns). TPS present in the microsomal membranes from rat cerebral cortex was used for these studies. Initial velocity measurements performed over a wide range of PAPS, BocCCK-8(ns), 3'-PAP and BocCCK-8(s) concentrations, indicated that the reaction follows an ordered mechanistic pathway. The KM value determined for BocCCK-8(ns) was 160 +/- 18 microM, and that for [35S]PAPS was 0.15 +/- 0.03 microM. 3'-Phosphoadenosine 5'-phosphate (3'-PAP) was found to be a product inhibitor with a Ki = 0.30 +/- 0.02 microM. BocCCK-8(s) produced an uncompetitive inhibition pattern on the TPS reaction. Adenosine 5'-phosphosulfate (APS) behaved as a competitive inhibitor versus PAPS with a Ki = 3.0 +/- 0.3 microM. ATP inhibited competitively the reaction when PAPS was the varied substrate with a Ki = 3.6 +/- 0.5 microM. The results of product and substrate inhibition studies and the patterns of dead end inhibition obtained with APS are best fit by an ordered Bi-Bi reaction mechanism where PAPS is the first substrate to bind and 3'-PAP is the last product to be released.     

Substrate recognition, protein dynamics, and iron-sulfur cluster in Pseudomonas aeruginosa adenosine 5'-phosphosulfate reductase

APS reductase catalyzes the first committed step of reductive sulfate assimilation in pathogenic bacteria, including Mycobacterium tuberculosis, and is a promising target for drug development. We report the 2.7 A resolution crystal structure of Pseudomonas aeruginosa APS reductase in the thiosulfonate intermediate form of the catalytic cycle and with substrate bound. The structure, high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, and quantitative kinetic analysis, establish that the two chemically discrete steps of the overall reaction take place at distinct sites on the enzyme, mediated via conformational flexibility of the C-terminal 18 residues. The results address the mechanism by which sulfonucleotide reductases protect the covalent but labile enzyme-intermediate before release of sulfite by the protein cofactor thioredoxin. P. aeruginosa APS reductase contains an [4Fe-4S] cluster that is essential for catalysis. The structure reveals an unusual mode of cluster coordination by tandem cysteine residues and suggests how this arrangement might facilitate conformational change and cluster interaction with the substrate. Assimilatory 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductases are evolutionarily related, homologous enzymes that catalyze the same overall reaction, but do so in the absence of an [Fe-S] cluster. The APS reductase structure reveals adaptive use of a phosphate-binding loop for recognition of the APS O3' hydroxyl group, or the PAPS 3'-phosphate group.     

Simple and fast purification of Escherichia coli adenylate kinase

Adenylate kinase from E. coli (strains CR341 and CR341 T28, a temperature-sensitive mutant) was purified by a two-step chromatographic procedure. The enzyme from crude extracts of both mutant and parent strain was bound to blue-Sepharose at pH 7.5, thereafter specifically eluted with 0.05 mM P1,P5-di(adenosine-5')pentaphosphate. A second chromatography on Sephadex G-100 yielded pure enzyme. E. coli adenylate kinase was strongly inhibited by P1,P5-di(adenosine-5')pentaphosphate (Ki 0.6 microM for adenylate kinase of strain CR341 and 2.1 microM in the case of mutant enzyme). After denaturation in 6 M guanidinium hydrochloride both mutant and parent adenylate kinase returned rapidly to the native, active state by dilution of guanidinium hydrochloride.     

Purification and properties of nucleotide pyrophosphatase from human placenta

Nucleotide pyrophosphatase was purified from human placenta to near homogeneity with a specific activity of about 500-fold over the Triton extract of the homogenate. Purification was achieved most effectively by successive chromatographic steps with AMP-agarose and ADP-agarose columns, based on the affinity of the enzyme towards 5'-adenylate and adenosine 3',5'-diphosphate, and a lectin-Sepharose column, based on the glycoprotein nature of the enzyme. The purified enzyme was found to be essentially homogeneous on SDS-polyacrylamide gel electrophoresis with a mobility corresponding to 130K. The purified enzyme was found to hydrolyze a wide variety of nucleotides, i.e. 3'-phosphoadenosine 5'-phosphosulfate (PAPS), adenosine 5'-phosphosulfate (APS), NADH, ATP, nucleotide sugars, oligonucleotides, and p-nitrophenyl-thymidine 5'-phosphate (PNTP). From the oligonucleotides, the enzyme produced 5'-phosphates. Mg2+ was required for full activity. Glycine and sulfhydryl compounds such as 2-mercaptoethanol and 2,3-dimercapto-1-propanol were inhibitory. Most of these properties are common to nucleotide pyrophosphatases [EC 3.6.1.9] and type I (5'-phosphate forming) phosphodiesterases [EC 3.1.4.1] from various sources. The relevance of this enzyme to a unique genetic disease, Lowe's syndrome, is discussed.