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.     

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 Penicillium chrysogenum: ligand binding properties and the mechanism of substrate inhibition.

[35S]Adenosine-5'-phosphosulfate (APS) binding to Penicillium chrysogenum APS kinase was measured by centrifugal ultrafiltration. APS did not bind to the free enzyme with a measurable affinity even at low ionic strength where substrate inhibition by APS is quite marked. However, APS bound with an apparent Kd of 0.54 microM in the presence of 5 mM MgADP. In the presence of 0.1 M (NH4)2SO4, Kd,app was increased to 2.1 +/- 0.7 microM. Bound [35S]APS was displaced by low concentrations of 3'-phosphoadenosine-5'-phosphosulfate (PAPS), or iso-(2') PAPS, or (less efficiently) by adenosine-3,5'-diphosphate (PAP) or adenosine-5'-monosulfate (AMS). The results support our conclusion that substrate inhibition of the fungal enzyme by APS results from the formation of a dead end E. MgADP.APS complex. That is, APS binds to the subsite vacated by PAPS in the compulsory (or predominately) ordered product release sequence (PAPS before MgADP). Radioligand displacement was used to verify the Kd for APS dissociation from E.MgADP.APS and to determine the Kd values for the dissociation of iso-PAPS (13 +/- 5 microM), PAP (4.8 mM), or AMS (5.2 mM) from their respective ternary enzyme.MgADP.ligand complexes. Incubation of the fungal enzyme with [gamma-32P]MgATP did not yield a phosphoenzyme that survives gel filtration or gel electrophoresis.     

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.     

Ligand-induced structural changes in adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum

Adenosine 5'-phosphosulfate (APS) kinase catalyzes the second reaction in the two-step, ATP-dependent conversion of inorganic sulfate to 3'-phosphoadenosine 5'-phosphosulfate (PAPS). PAPS serves as the sulfuryl donor for the biosynthesis of all sulfate esters and also as a precursor of reduced sulfur biomolecules in many organisms. Previously, we determined the crystal structure of ligand-free APS kinase from the filamentous fungus, Penicillium chrysogenum [MacRae et al. (2000) Biochemistry 39, 1613-1621]. That structure contained a protease-susceptible disordered region ("mobile lid"; residues 145-170). Addition of MgADP and APS, which together promote the formation of a nonproductive "dead-end" ternary complex, protected the lid from trypsin. This report presents the 1.43 A resolution crystal structure of APS kinase with both ADP and APS bound at the active site and the 2.0 A resolution structure of the enzyme with ADP alone bound. The mobile lid is ordered in both complexes and is shown to provide part of the binding site for APS. That site is formed primarily by the highly conserved Arg 66, Arg 80, and Phe 75 from the protein core and Phe 165 from the mobile lid. The two Phe residues straddle the adenine ring of bound APS. Arg 148, a completely conserved residue, is the only residue in the mobile lid that interacts directly with bound ADP. Ser 34, located in the apex of the P-loop, hydrogen-bonds to the 3'-OH of APS, the phosphoryl transfer target. The structure of the binary E.ADP complex revealed further changes in the active site and N-terminal helix that occur upon the binding/release of (P)APS.     

Structural changes of mitochondrial creatine kinase upon binding of ADP, ATP, or Pi, observed by reaction-induced infrared difference spectra

Structural modifications of rabbit heart mitochondrial creatine kinase induced by the binding of its nucleotide substrates and Pi were investigated. Reaction-induced difference spectra (RIDS), resulting from the difference between infrared spectra recorded before and after the photorelease of a caged ligand, allow us to detect very small variations in protein structure. Our results indicated that the protein secondary structure remained relatively stable during nucleotide binding. Indeed, this binding to creatine kinase affected only a few amino acids, and caused small peptide backbone deformations and alterations of the carbonyl side chains of aspartate or glutamate, reflecting modifications within preexisting elements rather than a net change in secondary structure. Nonetheless, MgADP and MgATP RIDS were distinct, whereas the MgPi RIDS presented some similarities with the MgATP one. The difference between MgADP and MgATP RIDS could reflect a distinct configuration of the two metal-nucleotide complexes inducing a different positioning and/or a distinct binding mode to the creatine kinase active site. Comparison of the MgATP and MgPi RIDS suggests that Pi binding took place at the same binding site as the gamma-phosphoryl group of ATP. Thus, the difference between MgADP and MgATP RIDS would mainly be due to the effect of the gamma-P of ATP. The differences observed when comparing the RIDS resulting from the binding of nucleotides to octameric mitochondrial creatine kinase or dimeric cytosolic isoform could reflect the distinct oligomerization states and physicochemical or kinetic properties of the two isoenzymes.     

Role of phosphate chain mobility of MgATP in completing the 3-phosphoglycerate kinase catalytic site: binding, kinetic, and crystallographic studies with ATP and MgATP

The complexes of pig muscle 3-phosphoglycerate kinase with the substrate MgATP and with the nonsubstrate Mg(2+)-free ATP have been characterized by binding, kinetic, and crystallographic studies. Comparative experiments with ADP and MgADP have also been carried out. In contrast to the less specific and largely ionic binding of Mg(2+)-free ATP and ADP, specific occupation of the adenosine binding pocket by MgATP and MgADP has been revealed by displacement experiments with adenosine and anions, as well as supported by isothermal calorimetric titrations. The Mg(2+)-free nucleotides similarly stabilize the overall protein structure and restrict the conformational flexibility around the reactive thiol groups of helix 13, as observed by differential scanning microcalorimetry and thiol reactivity studies, respectively. The metal complexes, however, behave differently. MgADP, but not MgATP, further increases the conformational stability with respect to its Mg(2+)-free form, which indicates their different modes of binding to the enzyme. Crystal structures of the binary complexes of the enzyme with MgATP and with ATP (2.1 and 1.9 A resolution, respectively) have shown that the orientation and interaction of phosphates of MgATP largely differ not only from those of ATP but also from the previously determined ones of either MgADP [Davies, G. J., Gamblin, S. J., Littlechild, J. A., Dauter, Z., Wilson, K. S., and Watson, H. C. (1994) Acta Crystallogr. D50, 202-209] or the metal complexes of AMP-PNP [May, A., Vas, M., Harlos, K., and Blake, C. C. F. (1996) Proteins 24, 292-303; Auerbach, G., Huber, R., Grattinger, M., Zaiss, K., Schurig, H., Jaenicke, R., and Jacob, U. (1997) Structure 5, 1475-1483] and are more similar to the interactions formed with MgAMP-PCP [Kovári, Z., Flachner, B., Náray-Szabó, G., and Vas, M. (2002) Biochemistry 41, 8796-8806]. Mg(2+) is liganded to both beta- and gamma-phosphates of ATP, while beta-phosphate is linked to the conserved Asp218, i.e., to the N-terminus of helix 8, through a water molecule; the known interactions of either MgADP or the metal complexes of AMP-PNP with the N-terminus of helix 13 and with Asn336 of beta-strand J are absent in the case of MgATP. Fluctuation of MgATP phosphates between two alternative sites has been proposed to facilitate the correct positioning of the mobile side chain of Lys215, and the catalytically competent active site is thereby completed.     

Mechanistic studies of Escherichia coli adenosine-5'-phosphosulfate kinase

Adenosine-5'-phosphosulfate kinase (APS kinase) catalyzes the formation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the major form of activated sulfate in biological systems. The enzyme from Escherichia coli has complex kinetic behavior, including substrate inhibition by APS and formation of a phosphorylated enzyme (E-P) as a reaction intermediate. The presence of a phosphorylated enzyme potentially enables the steady-state kinetic mechanism to change from sequential to ping-pong as the APS concentration decreases. Kinetic and equilibrium binding measurements have been used to evaluate the proposed mechanism. Equilibrium binding studies show that APS, PAPS, ADP, and the ATP analog AMPPNP each bind at a single site per subunit; thus, substrates can bind in either order. When ATPgammaS replaces ATP as substrate the V(max) is reduced 535-fold, the kinetic mechanism is sequential at each APS concentration, and substrate inhibition is not observed. The results indicate that substrate inhibition arises from a kinetic phenomenon in which product formation from ATP binding to the E. APS complex is much slower than paths in which product formation results from APS binding either to the E. ATP complex or to E-P. APS kinase requires divalent cations such as Mg(2+) or Mn(2+) for activity. APS kinase binds one Mn(2+) ion per subunit in the absence of substrates, consistent with the requirement for a divalent cation in the phosphorylation of APS by E-P. The affinity for Mn(2+) increases 23-fold when the enzyme is phosphorylated. Two Mn(2+) ions bind per subunit when both APS and the ATP analog AMPPNP are present, indicating a potential dual metal ion catalytic mechanism.     

Kinetic and stability properties of Penicillium chrysogenum ATP sulfurylase missing the C-terminal regulatory domain

ATP sulfurylase from Penicillium chrysogenum is a homohexameric enzyme that is subject to allosteric inhibition by 3'-phosphoadenosine 5'-phosphosulfate. In contrast to the wild type enzyme, recombinant ATP sulfurylase lacking the C-terminal allosteric domain was monomeric and noncooperative. All kcat values were decreased (the adenosine 5'-phosphosulfate (adenylylsulfate) (APS) synthesis reaction to 17% of the wild type value). Additionally, the Michaelis constants for MgATP and sulfate (or molybdate), the dissociation constant of E.APS, and the monovalent oxyanion dissociation constants of dead end E.MgATP.oxyanion complexes were all increased. APS release (the k6 step) was rate-limiting in the wild type enzyme. Without the C-terminal domain, the composite k5 step (isomerization of the central complex and MgPPi release) became rate-limiting. The cumulative results indicate that besides (a) serving as a receptor for the allosteric inhibitor, the C-terminal domain (b) stabilizes the hexameric structure and indirectly, individual subunits. Additionally, (c) the domain interacts with and perfects the catalytic site such that one or more steps following the formation of the binary E.MgATP and E.SO4(2-) complexes and preceding the release of MgPPi are optimized. The more negative entropy of activation of the truncated enzyme for APS synthesis is consistent with a role of the C-terminal domain in promoting the effective orientation of MgATP and sulfate at the active site.     

Characterization of the phosphorylated enzyme intermediate formed in the adenosine 5'-phosphosulfate kinase reaction.

Adenosine 5'-phosphosulfate (APS) kinase (ATP:APS 3'-phosphotransferase) catalyzes the ultimate step in the biosynthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the primary biological sulfuryl donor. APS kinase from Escherichia coli is phosphorylated upon incubation with ATP, yielding a protein that can complete the overall reaction through phosphorylation of APS. Rapid-quench kinetic experiments show that, in the absence of APS, ATP phosphorylates the enzyme with a rate constant of 46 s-1, which is equivalent to the Vmax for the overall APS kinase reaction. Similar pre-steady-state kinetic measurements show that the rate constant for transfer of the phosphoryl group from E-P to APS is 91 s-1. Thus, the phosphorylated enzyme is kinetically competent to be on the reaction path. In order to elucidate which amino acid residue is phosphorylated, and thus to define the active site region of APS kinase, we have determined the complete sequence of cysC, the structural gene for this enzyme in E. coli. The coding region contains 603 nucleotides and encodes a protein of 22,321 Da. Near the amino terminus is the sequence 35GLSGSGKS, which exemplifies a motif known to interact with the beta-phosphoryl group of purine nucleotides. The residue that is phosphorylated upon incubation with ATP has been identified as serine-109 on the basis of the amino acid composition of a radiolabeled peptide purified from a proteolytic digest of 32P-labeled enzyme. We have identified a sequence beginning at residue 147 which may reflect a PAPS binding site. This sequence was identified in the carboxy terminal region of 10 reported sequences of proteins of PAPS metabolism.     

Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5'-Phosphosulfate Kinase

Adenosine 5'-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3'-phosphate 5'-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp(136), which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis.     

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.     

Induction of positive cooperativity by amino acid replacements within the C-terminal domain of Penicillium chrysogenum ATP sulfurylase

ATP sulfurylase from Penicillium chrysogenum is an allosteric enzyme in which Cys-509 is critical for maintaining the R state. Cys-509 is located in a C-terminal domain that is 42% identical to the conserved core of adenosine 5'-phosphosulfate (adenylylsulfate) (APS) kinase. This domain is believed to provide the binding site for the allosteric effector, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Replacement of Cys-509 with either Tyr or Ser destabilizes the R state, resulting in an enzyme that is intrinsically cooperative at pH 8 in the absence of PAPS. The kinetics of C509Y resemble those of the wild type enzyme in which Cys-509 has been covalently modified. The kinetics of C509S resemble those of the wild type enzyme in the presence of PAPS. It is likely that the negative charge on the Cys-509 side chain helps to stabilize the R state. Treatment of the enzyme with a low level of trypsin results in cleavage at Lys-527, a residue that lies in a region analogous to a PAPS motif-containing mobile loop of true APS kinase. Both mutant enzymes were cleaved more rapidly than the wild type enzyme, suggesting that movement of the mobile loop occurs during the R to T transition.     

Elucidation of the active conformation of the APS-kinase domain of human PAPS synthetase 1

Bifunctional human PAPS synthetase (PAPSS) catalyzes, in a two-step process, the formation of the activated sulfate carrier 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The first reaction involves the formation of the 5'-adenosine phosphosulfate (APS) intermediate from ATP and inorganic sulfate. APS is then further phosphorylated on its 3'-hydroxyl group by an additional ATP molecule to generate PAPS. The former reaction is catalyzed by the ATP-sulfurylase domain and the latter by the APS-kinase domain. Here, we report the structure of the APS-kinase domain of PAPSS isoform 1 (PAPSS1) representing the Michaelis complex with the products ADP-Mg and PAPS. This structure provides a rare glimpse of the active conformation of an enzyme catalyzing phosphoryl transfer without resorting to substrate analogs, inactivating mutations, or catalytically non-competent conditions. Our structure shows the interactions involved in the binding of the magnesium ion and PAPS, thereby revealing residues critical for catalysis. The essential magnesium ion is observed bridging the phosphate groups of the products. This function of the metal ion is made possible by the DGDN-loop changing its conformation from that previously reported, and identifies these loop residues unambiguously as a Walker B motif. Furthermore, the second aspartate residue of this motif is the likely candidate for initiating nucleophilic attack on the ATP gamma-phosphate group by abstracting the proton from the 3'-hydroxyl group of the substrate APS. We report the structure of the APS-kinase domain of human PAPSS1 in complex with two APS molecules, demonstrating the ability of the ATP/ADP-binding site to bind APS. Both structures reveal extended N termini that approach the active site of the neighboring monomer. Together, these results significantly increase our understandings of how catalysis is achieved by APS-kinase.     

Crystal structure of the bifunctional ATP sulfurylase-APS kinase from the chemolithotrophic thermophile Aquifex aeolicus

The thermophilic chemolithotroph, Aquifex aeolicus, expresses a gene product that exhibits both ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase activities. These enzymes are usually segregated on two separate proteins in most bacteria, fungi, and plants. The domain arrangement in the Aquifex enzyme is reminiscent of the fungal ATP sulfurylase, which contains a C-terminal domain that is homologous to APS kinase yet displays no kinase activity. Rather, in the fungal enzyme, the motif serves as a sulfurylase regulatory domain that binds the allosteric effector 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the product of true APS kinase. Therefore, the Aquifex enzyme may represent an ancestral homolog of a primitive bifunctional enzyme, from which the fungal ATP sulfurylase may have evolved. In heterotrophic sulfur-assimilating organisms such as fungi, ATP sulfurylase catalyzes the first committed step in sulfate assimilation to produce APS, which is subsequently metabolized to generate all sulfur-containing biomolecules. In contrast, ATP sulfurylase in sulfur chemolithotrophs catalyzes the reverse reaction to produce ATP and sulfate from APS and pyrophosphate. Here, the 2.3 A resolution X-ray crystal structure of Aquifex ATP sulfurylase-APS kinase bifunctional enzyme is presented. The protein dimerizes through its APS kinase domain and contains ADP bound in all four active sites. Comparison of the Aquifex ATP sulfurylase active site with those from sulfate assimilators reveals similar dispositions of the bound nucleotide and nearby residues. This suggests that minor perturbations are responsible for optimizing the kinetic properties for the physiologically relevant direction. The APS kinase active-site lid adopts two distinct conformations, where one conformation is distorted by crystal contacts. Additionally, a disulfide bond is observed in one ATP-binding P-loop of the APS kinase active site. This linkage accounts for the low kinase activity of the enzyme under oxidizing conditions. The thermal stability of the Aquifex enzyme can be explained by the 43% decreased cavity volume found within the protein core.     

Crystal structure of human riboflavin kinase reveals a beta barrel fold and a novel active site arch

Riboflavin kinase (RFK) is an essential enzyme catalyzing the phosphorylation of riboflavin (vitamin B(2)) to form FMN, an obligatory step in vitamin B(2) utilization and flavin cofactor synthesis. The structure of human RFK revealed a six-stranded antiparallel beta barrel core structurally similar to the riboflavin synthase/ferredoxin reductase FAD binding domain fold. The binding site of an intrinsically bound MgADP defines a novel nucleotide binding motif that encompasses a loop, a 3(10) helix, and a reverse turn followed by a short beta strand. This active site loop forms an arch with ATP and riboflavin binding at the opposite side and the phosphoryl transfer appears to occur through the hole underneath the arch. The invariant residues Asn36 and Glu86 are implicated in the catalysis.     

Loop movement and catalysis in creatine kinase

Recently the crystal structure of creatine kinase from Torpedocalifornica was determined to 2.1 A. The dimeric structure revealed two different forms in the unit cell: one monomer was bound to a substrate, MgADP, and the other monomer was bound to a transition-state analogue complex composed of MgADP, nitrate and creatine. The most striking difference between the structures is the movement of two loops (comprising residues 60-70 and residues 323-333) into the active site in the transition state structure. This loop movement effectively occludes the active site from solvent, and the loops appear to be locked into place by a salt bridge formed between His66 and Asp326. His66 is of particular interest as it is located within a PGHP motif conserved in all creatine kinases but not found in other guanidino kinases. We have carried out alanine-scanning mutagenesis of each of the residues in the PGHP motif and determined that only the His66 plays a significant role in the creatine kinase reaction. Although neither residue interacts directly with the substrate, the interaction His66 and Asp326 appears to be important in providing the precise alignment of substrates necessary for phosphoryl group transfer. Finally, it is clear that neither His66 nor Asp326 are responsible for the pKs observed in the pH-rate profile for HMCK.     

Persistent binding of MgADP to the E187A mutant of Escherichia coli phosphofructokinase in the absence of allosteric effects

MgADP binding to the allosteric site enhances the affinity of Escherichia coli phosphofructokinase (PFK) for fructose 6-phosphate (Fru-6-P). X-ray crystallographic data indicate that MgADP interacts with the conserved glutamate at position 187 within the allosteric site through an octahedrally coordinated Mg(2+) ion [Shirakihara, Y., and Evans, P. R. (1988) J. Mol. Biol. 204, 973-994]. Lau and Fersht reported that substituting an alanine for this glutamate within the allosteric site of PFK (i.e., mutant E187A) causes MgADP to lose its allosteric effect upon Fru-6-P binding [Lau, F. T.-K., and Fersht, A. R. (1987) Nature 326, 811-812]. However, these authors later reported that MgADP inhibits Fru-6-P binding in the E187A mutant. The inhibition presumably occurs by preferential binding to the inactive (T) state complex of the Monod-Wyman-Changeux two-state model [Lau, F. T.-K., and Fersht, A. R. (1989) Biochemistry 28, 6841-6847]. The present study provides an alternative explanation of the role of MgADP in the E187A mutant. Using enzyme kinetics, steady-state fluorescence emission, and anisotropy, we performed a systematic linkage analysis of the three-ligand interaction between MgADP, Fru-6-P, and MgATP. We found that MgADP at low concentrations did not enhance or inhibit substrate binding. Anisotropy shows that MgADP binding at the allosteric site occurred even when MgADP produced no allosteric effect. However, as in the wild-type enzyme, the binding of MgADP to the active site in the mutant competitively inhibited MgATP binding and noncompetitively inhibited Fru-6-P binding. These results clarified the mechanism of a three-ligand interaction and offered a nontraditional perspective on allosteric mechanism.     

Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly

Combined mutation of "catalytic carboxylates" in both nucleotide binding domains (NBDs) of P-glycoprotein generates a conformation capable of tight binding of 8-azido-ADP (Sauna, Z. E., Müller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry 41, 13989-14000). Here we characterized this conformation using pure mouse MDR3 P-glycoprotein and natural MgATP and MgADP. Mutants E552A/E1197A, E552Q/E1197Q, E552D/E1197D, and E552K/E1197K had low but real ATPase activity in the order Ala > Gln > Asp > Lys, emphasizing the requirement for Glu stereochemistry. Mutant E552A/E1197A bound MgATP and MgADP (1 mol/mol) with K(d) 9.2 and 92 microm, showed strong temperature sensitivity of MgATP binding and equal dissociation rates for MgATP and MgADP. With MgATP as the added ligand, 80% of bound nucleotide was in the form of ATP. None of these parameters was vanadate-sensitive. The other mutants showed lower stoichiometry of MgATP and MgADP binding, in the order Ala > Gln > Asp > Lys. We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized NBD dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly. Impairment of turnover is primarily due to inability to form the normal transition state rather than to slow ADP release. The Gln, Asp, and Lys mutants are less effective at stabilizing the occluded nucleotide, putative dimeric NBD, conformation. We envisage that in wild-type the occluded nucleotide conformation occurs transiently after MgATP binds to both NBDs with associated dimerization, and before progression to the transition state.     

Binding of allosteric effectors to carbamyl-phosphate synthetase from Escherichia coli.

The binding of ornithine and inosine 5'-monophosphate (IMP), positive allosteric effectors, and of uridine 5'-monophosphate (UMP), a negative allosteric effector, to carbamyl-phosphate synthetase from Escherichia coli was studied by the technique of equilibrium dialysis. The monomeric form of the enzyme has one binding site for each of the three allosteric ligands. The binding of UMP is inhibited by ornithine, IMP, MgATP, and ammonia (also a positive allosteric effector). Bicarbonate, L-glutamine, and adenosine 5'-triphosphate (ATP) (Mg2+ absent) had no effect on the binding of UMP. The affinity of the enzyme for UMP was increased if phosphate buffer was replaced by 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) buffer. The binding of ornithine was inhibited by UMP and ammonia, enhanced by MgATP, MgADP, and IMP, and not affected by bicarbonate, L-glutamine, or ATP (Mg2+ absent). Ornithine and ammonia probably bind to the same site on the enzyme. The binding of IMP is facilitated by ornithine and ammonia, but is inhibited by MgATP or ATP, indicating that adenine nucleotides can also bind to the IMP binding site. The results of these binding studies are consistent with a scheme previously proposed in which the allosteric effectors function by stabilizing one or the other of two different conformational states of the enzyme which are in equilibrium with each other (Anderson, P.M., and Marvin, S.V. (1970), Biochemistry 9, 171). According to this scheme, binding of the substrate MgATP is greatly facilitated when the enzyme exists in the conformational state stabilized by the positive allosteric effectors.