Kinetic and spectroscopic studies of the ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri: substrate specificity and insights into the mechanism of Co(II)corrinoid reduction

The PduO-type ATP:corrinoid adenosyltransferase from Lactobacillus reuteri ( LrPduO) catalyzes the formation of the essential Co-C bond of adenosylcobalamin (coenzyme B 12) by transferring the adenosyl group from cosubstrate ATP to a transient Co (1+)corrinoid species generated in the enzyme active site. While PduO-type enzymes have previously been believed to be capable of adenosylating only Co (1+)cobalamin (Co (1+)Cbl (-)), our kinetic data obtained in this study provide in vitro evidence that LrPduO can in fact also utilize the incomplete corrinoid Co (1+)cobinamide (Co (1+)Cbi) as an alternative substrate. To explore the mechanism by which LrPduO overcomes the thermodynamically challenging reduction of its Co (2+)corrinoid substrates, we have examined how the enzyme active site alters the geometric and electronic properties of Co (2+)Cbl and Co (2+)Cbi (+) by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopic techniques. Our data reveal that upon binding to LrPduO that was preincubated with ATP, both Co (2+)corrinoids undergo a partial ( approximately 40-50%) conversion to distinct paramagnetic Co (2+) species. The spectroscopic signatures of these species are consistent with essentially four-coordinate, square-planar Co (2+) complexes, based on a comparison with the results obtained in our previous studies of related enzymes. Consequently, it appears that the general strategy employed by adenosyltransferases for effecting Co (2+) --> Co (1+) reduction involves the formation of an "activated" Co (2+)corrinoid intermediate that lacks any significant axial bonding interactions, to stabilize the redox-active, Co 3d z (2) -based molecular orbital.     

Effects of single nucleotide changes on the binding and activity of RNA aptamers to human papillomavirus 16 E7 oncoprotein

ATP:Cobalamin adenosyltransferases catalyze the transfer a 5′-deoxyadenosyl moiety from ATP to cob(I)alamin in the synthesis of the Co–C bond of coenzyme B₁₂. There are three types of adenosyltransferases, CobA, PduO and EutT. Among these adenosyltransferases, the PduO-type adenosyltransferases is the most widely distributed enzyme. Structural comparisons between apo BcPduO and BcPduO in complex with MgATP revealed that the N-terminal strands of both structures were ordered, which is in contrast with the most previously available PduO-type adenosyltransferase structures. Furthermore, unlike other reported structures, apo BcPduO was bound to additional dioxane molecules causing a side chain conformational change at the Tyr30 residue, which is an important residue that mediates hydrogen bonding with ATP molecules upon binding of cobalamin to the active site. This study provides more structural information into the role of active site residues on substrate binding.     

Purification and initial characterization of the ATP:corrinoid adenosyltransferase encoded by the cobA gene of Salmonella typhimurium.

The cobA gene of Salmonella typhimurium and its product were overexpressed to approximately 20% of the total cell protein. CobA was purified to 98% homogeneity; N-terminal sequence analysis (21 residues) of homogeneous protein confirmed the predicted amino acid sequence. ATP:corrinoid adenosyltransferase activity was demonstrated in vitro to be associated with CobA. This activity was optimal at pH 8 and 37 degrees C. A quantitative preference was determined for Mn(II) cations and ATP. The apparent Km of CobA for ATP was 2.8 microM, and that for cob(I)alamin was 5.2 microM. Vmax was measured at 0.43 nmol/min. Cobinamide served as the substrate for CobA to yield adenosylcobinamide. Activity was stable at 4 degrees C for several weeks but was lost rapidly at room temperature (50% overnight). Dithiothreitol was required to maintain the enzymatic activity of CobA.     

Structural and functional analyses of the human-type corrinoid adenosyltransferase (PduO) from Lactobacillus reuteri

ATP:Co(I)rrinoid adenosyltransferase (ACA) catalyzes the conversion of cobalamin to coenzyme B12, an essential cofactor in animal metabolism. Several mutations of conserved residues in the active site of human ACA have been identified in humans with methylmalonic aciduria. However, the catalytic role of these residues remains unclear. To better understand the function of these residues and to determine how the enzyme promotes catalysis, several variants of a human-type ACA from the lactic acid bacterium Lactobacillus reuteri (LrPduO) were kinetically and structurally characterized. Kinetic analyses of a series of alternate nucleotides were also performed. Substrate inhibition was observed at subsaturating concentrations of ATP, consistent with an ordered binding scheme where ATP is bound first by the enzyme. Modification or elimination of an active site, inter-subunit salt bridge resulted in a reduced "on" rate for ATP binding, with a less significant disruption in the rate of subsequent catalytic steps. Kinetic and structural data demonstrate that residue Arg132 is not involved in orienting ATP in the active site but, rather, it stabilizes the altered substrate in the transition state. Two functional groups of ATP explain the reduced ability of the enzyme to use alternate nucleotides: the amino group at the C-6 position of ATP contributes approximately 6 kcal/mol of free energy to ground state binding, and the nitrogen at the N-7 position assists in coordinating the magnesium ion in the active site. This study provides new insight into the role of substrate binding determinants and active site residues in the reaction catalyzed by a human-type ACA.     

Dihydroflavin-driven Adenosylation of 4-Coordinate Co(II) Corrinoids: ARE COBALAMIN REDUCTASES ENZYMES OR ELECTRON TRANSFER PROTEINS?*

The identity of the source of the biological reductant needed to convert cobalamin to its biologically active form adenosylcobalamin has remained elusive. Here we show that free or protein-bound dihydroflavins can serve as the reductant of Co²⁺Cbl bound in the active site of PduO-type ATP-dependent corrinoid adenosyltransferase enzymes. Free dihydroflavins (dihydroriboflavin, FMNH₂, and FADH₂) effectively drove the adenosylation of Co²⁺Cbl by the human and bacterial PduO-type enzymes at very low concentrations (1 μm). These data show that adenosyltransferase enzymes lower the thermodynamic barrier of the Co²⁺ → Co⁺ reduction needed for the formation of the unique organometalic Co–C bond of adenosylcobalamin. Collectively, our in vivo and in vitro data suggest that cobalamin reductases identified thus far are most likely electron transfer proteins, not enzymes.     

Molecular properties of two proteins homologous to PduO-type ATP:cob(I)alamin adenosyltransferase from Sulfolobus tokodaii

In the thermophilic archaeon Sulfolobus tokodaii, there are two genes homologous to PduO-type ATP:cob(I)alamin adenosyltransferase, ST1454 and ST2180. To address the structure and function of these two sequence-related proteins from one organism, we prepared them by using the Escherichia coli expression system and analyzed them by immunoblotting, matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy, circular dichroism spectrometry, ATP:cobalamin adenosyltransferase assay, and X-ray crystallography. Immunoblotting and matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy analyses showed that both these proteins are expressed in S. tokodaii cells as soluble proteins and are spontaneously digested at the N-terminal region. ATP:cob(I)alamin adenosyltransferase activity was detected for ST1454 but not for ST2180. ST2180 reduced the concentration of cob(I)alamin, suggesting that ST2180 might recognize cob(I)alamin as a ligand. The secondary structure of ST1454 was retained even in 7 M guanidine hydrochroride, whereas that of ST2180 was melted in 4.5 M guanidine hydrochloride. The X-ray crystal structural analysis revealed that the proteins shared a common structure: a trimer of five-helix bundles with a clockwise kink. There is a pocket surrounded by highly conserved residues, in which a polypropylene glycol 400 in the crystal structure of ST1454 was captured, suggesting that it is an active site. Structural comparison between these two proteins showed the difference in the number of ion pairs around the proposed active site. On the basis of these results, we propose that ST1454 and ST2180 have related but distinct functions.     

Purification and initial characterization of the Salmonella enterica PduO ATP:Cob(I)alamin adenosyltransferase

The PduO enzyme of Salmonella enterica is an ATP:cob(I)alamin adenosyltransferase that catalyzes the final step in the conversion of vitamin B(12) to coenzyme B(12). The primary physiological role of this enzyme is to support coenzyme B(12)-dependent 1,2-propanediol degradation, and bioinformatic analysis has indicated that it has two domains. Here the PduO adenosyltransferase was produced in Escherichia coli, solubilized from inclusion bodies, purified to apparent homogeneity, and partially characterized biochemically. The K(m) values of PduO for ATP and cob(I)alamin were 19.8 and 4.5 microM, respectively, and the enzyme V(max) was 243 nmol min(-1) mg of protein(-1). Further investigations showed that PduO was active with ATP and partially active with deoxy-ATP, but lacked measurable activity with other nucleotides. (31)P nuclear magnetic resonance established that triphosphate was a product of the PduO reaction, and kinetic studies indicated a ternary complex mechanism. A series of truncated versions of the PduO protein were produced in Escherichia coli, partially purified, and used to show that adenosyltransferase activity is associated with the N-terminal domain. The N-terminal domain was purified to near homogeneity and shown to have biochemical properties and kinetic constants similar to those of the full-length enzyme. This indicated that the C-terminal domain was not directly involved in catalysis or substrate binding and may have another role.     

The structural basis for methylmalonic aciduria. The crystal structure of archaeal ATP:cobalamin adenosyltransferase

ATP:cobalamin adenosyltransferase MMAB was recently identified as the gene responsible for a disorder of cobalamin metabolism in humans (cblB complementation group). The crystal structure of the MMAB sequence homologue from Thermoplasma acidophilum (TA1434; GenBank identification number gi|16082403) was determined to a resolution of 1.5 A. TA1434 was confirmed to be an ATP:cobalamin adenosyltransferase, which depended absolutely on divalent metal ions (Mg2+ > Mn2+ > Co2+) and only used ATP or dATP as adenosyl donors. The apparent Km of TA1434 was 110 microM (kcat = 0.23 s(-1)) for ATP, 140 microM (kcat = 0.11 s(-1)) for dATP, and 3 microM (kcat = 0.18 s(-1)) for cobalamin. TA1434 is a trimer in solution and in the crystal structure, with each subunit composed of a five-helix bundle. The location of disease-related point mutations and other residues conserved among the homologues of TA1434 suggest that the active site lies at the junctions between the subunits. Mutations in TA1434 that correspond to the disease-related mutations resulted in proteins that were inactive for ATP:cobalamin adenosyltransferase activity in vitro, confirming that these mutations define the molecular basis of the human disease.     

Dual functional roles of ATP in the human mitochondrial malic enzyme

Human mitochondrial malic enzyme is a regulatory enzyme with ATP as an inhibitor. Structural studies reveal that the enzyme has two ATP-binding sites, one at the NAD(+)-binding site in the active center and the other at the exo site in the tetramer interface. Inhibition of the enzyme activity is due to the competition between ATP and NAD(+) for the nucleotide-binding site at the active center with an inhibition constant of 81 microM. Binding of the ATP molecule at the exo site, on the other hand, is important for the maintenance of the quaternary structural integrity. The enzyme exists in solution at neutral pH and at equilibrium of the dimer and tetramer with a dissociation constant (K(TD)) of 0.67 microM. ATP, at a physiological concentration, shifts the equilibrium toward tetramer and decreases the K(TD) by many orders of magnitude. Mutation of a single residue Arg542 at the tetrameric interfacial exo site resulted in dimeric mutants. ATP thus has dual functional roles in the mitochondrial malic enzyme.     

Kinetic characterization of the carbon monoxide-acetyl-CoA (carbonyl group) exchange activity of the acetyl-CoA synthesizing CO dehydrogenase from Clostridium thermoaceticum

CO dehydrogenase from Clostridium thermoaceticum is a nickel-containing enzyme that catalyzes both the reversible conversion of CO2 to CO (for incorporation into the carbonyl group of acetate) and the synthesis of acetyl-CoA from methyl corrinoid, CO, and CoASH. The latter activity is conveniently assayed by monitoring the exchange of [1-14C]acetyl-CoA (carbonyl group) with 12CO. Kinetic parameters for the highly oxygen sensitive exchange activity have been determined: Km (acetyl-CoA) = 600 microM; Vmax = 440 min-1. In addition, coenzyme A analogues have been tested as inhibitors of the exchange to probe the active site of the enzyme; each has no effect on the CO2 in equilibrium CO activity of CO dehydrogenase. Coenzyme A, the substrate for acetate biosynthesis, is a potent competitive inhibitor, KI = 7 microM. Comparison of this value with that for desulfo-CoA (KI = 6000 microM) suggests that a key mode of binding is through the sulfur atom, possibly to a metal site on the enzyme. The relatively high affinity of the enzyme for CoASH relative to acetyl-CoA is consistent with its proposed operation in the acetogenic direction. The differential sensitivity to oxygen and storage of the two activities of CO dehydrogenase as well as the contrasting effect of coenzyme A inhibitors suggests that acetate assemblage occurs at a site distinct from that for CO dehydrogenation.     

Characterization of the high and low affinity components of the renal Ca2(+)-Mg2+ ATPase

The purpose of this study was to characterize the interrelationship between free calcium (Ca2+) and magnesium (Mg2+) in the Ca2+ ATPase enzyme cycle of kidney membranes. Experiments were performed with basolateral membranes from rat renal cortex and microdissected proximal and distal tubules from mice. Results were similar in the three types of preparations. We first investigated the effect of ATP concentration on Ca2(+)- and Mg2(+)-dependent ATP hydrolysis. With 0.2 microM Ca2+, the enzyme activity, as a function of ATP concentration, showed two saturable components: a high affinity component with a Km of 33 microM ATP and a low affinity component with a Km of 0.63 mM ATP. These components may represent either two distinct sites of ATP binding or two forms of the same site. For the sake of simplicity, it was assumed that the two components correspond to a high affinity and a low affinity substrate site. At the high affinity site (ATP = 50 microM), the Ca2+ dependence of ATP hydrolysis followed a single Michaelis-Menten kinetics with Km for Ca2+ of 0.08 microM. The addition of 1 mM Mg2+ resulted in a relatively constant increase in ATP hydrolysis at all Ca2+ concentrations, indicating that the effects of the two cations were additive. With high ATP concentration (ATP = 3 mM), Ca2+ also induced an ATP hydrolysis according to a saturable process, with a Km for Ca2+ of 0.2 microM. In contrast with what occurred with low concentrations of ATP, addition of millimolar Mg2+ completely curtailed the sensitivity of the enzyme to Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of the CobA-type ATP:Co(I)rrinoid adenosyltransferase enzyme of Methanosarcina mazei strain Go1

Although methanogenic archaea use B(12) extensively as a methyl carrier for methanogenesis, little is known about B(12) metabolism in these prokaryotes or any other archaea. To improve our understanding of how B(12) metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain G?1 (open reading frame MM3138, referred to as cobA(Mm) here) was cloned and used to restore coenzyme B(12) synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme. cobA(Mm) protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca(2+) ions, but the activity was not enhanced by Mg(2+) and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn(2+). The CobA(Mm) enzyme had a K(m)(ATP) of 3 microM and a K(m)(HOCbl) of 1 microM. Unlike the S. enterica enzyme, CobA(Mm) used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the beta phosphate of ATP was required for binding to the enzyme. A striking difference between CobA(Se) and CobA(Mm) was the use of ADP as a substrate by CobA(Mm), suggesting an important role for the gamma phosphate of ATP in binding. The results from (31)P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPP(i)) is the reaction by-product; no cleavage of PPP(i) was observed, and the enzyme was only slightly inhibited by pyrophosphate (PP(i)). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobA(Se) and CobA(Mm) enzymes.     

The ATP:Co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica requires the 2'-OH group of ATP for function and yields inorganic triphosphate as its reaction byproduct

The specificity of the ATP:corrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica serovar Typhimurium LT2 for its nucleotide substrate was tested using ATP analogs and alternative nucleotide donors. The enzyme showed broad specificity for the nucleotide base and required the 2'-OH group of the ribosyl moiety of ATP for activity. (31)P NMR spectroscopy was used to identify inorganic triphosphate (PPP(i)) as the byproduct of the reaction catalyzed by the CobA enzyme. Cleavage of triphosphate into pyrophosphate and orthophosphate did not occur, indicating that triphosphate cleavage was not required for release of the adenosylcorrinoid product. Triphosphate was a strong inhibitor of the reaction, with 85% of CobA activity lost when the ATP/PPP(i) ratio present in the reaction mixture was 1:2.5.     

Investigation of the catalytic site within the ATP-grasp domain of Clostridium symbiosum pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, P(i), and pyruvate with AMP, PP(i), and phosphoenolpyruvate (PEP) in three partial reactions as follows: 1) E-His + ATP --> E-His-PP.AMP; 2) E-His-PP.AMP + P(i) --> E-His-P.AMP.PP(i); and 3) E-His-P + pyruvate --> E.PEP using His-455 as the carrier of the transferred phosphoryl groups. The crystal structure of the Clostridium symbiosum PPDK (in the unbound state) reveals a three-domain structure consisting of consecutive N-terminal, central His-455, and C-terminal domains. The N-terminal and central His-455 domains catalyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3. Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide binding domain have been unsuccessful. The object of the present study is to demonstrate Mg(II) activation of catalysis at the ATP/P(i) active site, to identify the residues at the ATP/P(i) active site that contribute to catalysis, and to identify roles for these residues based on their positions within the active site scaffold. First, Mg(II) activation studies of catalysis of E + ATP + P(i) --> E-P + AMP + PP(i) partial reaction were carried out using a truncation mutant (Tem533) in which the C-terminal domain is absent. The kinetics show that a minimum of 2 Mg(II) per active site is required for the reaction. The active site residues used for substrate/cofactor binding/activation were identified by site-directed mutagenesis. Lys-22, Arg-92, Asp-321, Glu-323, and Gln-335 mutants were found to be inactive; Arg-337, Glu-279, Asp-280, and Arg-135 mutants were partially active; and Thr-253 and Gln-240 mutants were almost fully active. The participation of the nucleotide ribose 2'-OH and alpha-P in enzyme binding is indicated by the loss of productive binding seen with substrate analogs modified at these positions. The ATP, P(i), and Mg(II) ions were docked into the PPDK N-terminal domain crevice, in an orientation consistent with substrate/cofactor binding modes observed for other members of the ATP-Grasp fold enzyme superfamily and consistent with the structure-function data. On the basis of this docking model, the ATP polyphosphate moiety is oriented/activated for pyrophosphoryl transfer through interaction with Lys-22 (gamma-P), Arg-92 (alpha-P), and the Gly-101 to Met-103 loop (gamma-P) as well as with the Mg(II) cofactors. The P(i) is oriented/activated for partial reaction 2 through interaction with Arg-337 and a Mg(II) cofactor. The Mg(II) ions are bound through interaction with Asp-321, Glu-323, and Gln-335 and substrate. Residues Glu-279, Asp-280, and Arg-135 are suggested to function in the closure of an active site loop, over the nucleotide ribose-binding site.     

High Km soluble 5'-nucleotidase from human placenta. Properties and allosteric regulation by IMP and ATP

A human placental soluble "high Km" 5'-nucleotidase has been separated from "low Km" 5'-nucleotidase and nonspecific phosphatase by AMP-Sepharose affinity chromatography. The enzyme was purified 8000-fold to a specific activity of 25.6 mumol/min/mg. The subunit molecular mass is 53 kDa, and the native molecular mass is 210 kDa, suggesting a tetrameric structure. Soluble high Km 5'-nucleotidase is most active with IMP and GMP and their deoxy derivatives. IMP is hydrolyzed 15 times faster than AMP. The enzyme has a virtually absolute requirement for magnesium ions and is regulated by them. Purine nucleoside 5'-triphosphates strongly activate the enzyme with the potency order dATP greater than ATP greater than GTP. 2,3-Diphosphoglycerate activates the enzyme as potently as ATP. Three millimolar ATP decreased the Km for IMP from 0.33 to 0.09 mM and increased the Vmax 12-fold. ATP activation was modified by the IMP concentration. At 20 microM IMP the ATP-dependent activation curve was sigmoidal, while at 2 mM IMP it was hyperbolic. The A0.5 values for ATP were 2.26 and 0.70 mM, and the relative maximal velocities were 32.9 and 126.0 nmol/min, respectively. Inorganic phosphate shifts the hyperbolic substrate velocity relationship for IMP to a sigmoidal one. With physiological concentrations of cofactors (3 mM ATP, 1-4 mM Pi, 150 mM KCl) at pH 7.4, the enzyme is 25-35 times more active toward 100 microM IMP than 100 microM AMP. These data show that: (a) soluble human placental high Km 5'-nucleotidase coexists in human placenta with the low Km enzyme; (b) under physiological conditions the enzyme favors the hydrolysis of IMP and is critically regulated by IMP, ATP, and Pi levels; and (c) kinetic properties of ATP and IMP are each modified by the other compound suggesting complex interaction of the associated binding sites.     

Kinetics of the acid pump in the stomach. Proton transport and hydrolysis of ATP and p-nitrophenyl phosphate by the gastric H,K-ATPase

Hydrolysis of adenosine 5'-triphosphate (ATP) and p-nitrophenyl phosphate by the hydrogen ion-transporting potassium-stimulated adenosine triphosphatase (H,K-ATPase) was investigated. Hydrolysis of ATP was studied at pH 7.4 in vesicles treated with the ionophore nigericin. The kinetic analysis showed negative cooperativity with one high affinity (Km1 = 3 microM) and one low affinity (Km2 = 208 microM) site for ATP. The rate of hydrolysis decreased at 2000 microM ATP indicating a third site for ATP. When the pH was decreased to 6.5 the experimental results followed Michaelis-Menten enzyme kinetics with one low affinity site (Km = 116 microM). Higher concentrations than 750 microM ATP were inhibitory. Proton transport was measured as accumulation of acridine orange in vesicles equilibrated with 150 mM KCl. The transport at various concentrations of ATP in the pH interval from 6.0 to 8.0 correlated well with the Hill equation with a Hill coefficient between 1.5-1.9. The concentration of ATP resulting in half-maximal transport rate (S0.5) increased from 5 microM at pH 6.0 to 420 microM at pH 8.0. At acidic pH the rate of proton transport decreased at 1000 microM ATP. The K+-stimulated p-nitrophenylphosphatase (pNPPase) activity resulted in a Hill coefficient close to 2 indicating cooperative binding of substrate. The pNPPase was noncompetitively inhibited by ATP and ADP; half-maximal inhibition was obtained at 2 and 100 microM, respectively. Phospholipase C-treated vesicles lost 80% of the pNPPase activity, but the Hill coefficient did not change. These kinetic results are used for a further development of the reaction scheme of the H,K-ATPase.     

Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

The prsA1 allele, specifying a mutant Escherichia coli phosphoribosylpyrophosphate (PRPP) synthetase, has been cloned. The mutation was shown by nucleotide sequence analysis to result from substitution of Asp-128 (GAT) in the wild type by Ala (GCT) in prsA1. This alteration was confirmed by chemical determination of the amino acid sequence of a tryptic peptide derived from the purified mutant enzyme. The mutation lies at the N-terminal end of a 16 residue sequence that is highly conserved in E. coli, Bacillus subtilis, and rat PRPP synthetases and has the following consensus sequence: DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6-fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent cation binds to PRPP synthetase and serves as a bridge to the alpha-phosphate of ATP and AMP at the active site. The prsA1 mutation appears to alter this divalent cation site.     

Novel effects of calmodulin and calmodulin antagonists on the plasma membrane (Ca2+ + Mg2+)-ATPase from rabbit kidney proximal tubules.

In this work we report an unusual pattern of activation by calmodulin on the (Ca2+ + Mg2+)-ATPase from basolateral membranes of kidney proximal tubule cells. The activity of the ATPase depleted of calmodulin is characterized by a high Ca2+ affinity (Km = 2.2-3.4 microM) and a biphasic dependence on ATP concentration. The preparation responded to the addition of calmodulin by giving rise to a new Ca2+ site of very high affinity (Km less than 0.05 microM). Calmodulin antagonists had diverse effects on ATPase activity. Compound 48/80 inhibited calmodulin-stimulated activity by 70%, whereas calmidazolium did not modify this component. In the absence of calmodulin, 48/80 still acted as an antagonist, increasing the Km for Ca2+ to 5.7 microM and reducing enzyme turnover by competing with ATP at the low affinity regulatory site. Calmidazolium did not affect Ca2+ affinity, but it did displace ATP from the regulatory site. At fixed Ca2+ (30 microM) and ATP (5 mM) concentrations, Pi protected against 48/80 and potentiated inhibition by calmidazolium. At 25 microM ATP, Pi protected against calmidazolium inhibition. We propose that the effects of ATP and Pi arise because binding of the drugs to the ATPase occurs mainly on the E2 forms.     

Purification and characterization of delta helicase from fetal calf thymus.

A DNA helicase (delta helicase) which partially copurifies with DNA polymerase delta has been highly purified from fetal calf thymus. delta helicase differs in physical and enzymatic properties from other eukaryotic DNA helicases described thus far. The enzyme has an apparent mass of 57 kDa by gel filtration and is associated with polypeptides of 56 and 52 kDa by SDS-polyacrylamide gel electrophoresis. Photo-cross-linking of the purified enzyme with [alpha-32P]ATP resulted in labeling of a polypeptide of approximately 58 kDa, suggesting that the active site is present on the larger polypeptide. Unwinding of a partial duplex requires a nucleoside triphosphate which can be either ATP or dATP but not a nonhydrolyzable analogue of ATP. Other ribo- and deoxyribonucleoside triphosphates have little or no activity as cofactors. delta helicase also has DNA-dependent ATPase activity which has a relatively low Km for ATP (40 microM). delta helicase binds to single-stranded DNA but has little or no affinity for double-stranded DNA or single-stranded RNA. Similar to replicative DNA helicases from prokaryotes and the herpes simplex virus type 1 helicase-primase, delta helicase translocates in the 5'-3' direction along the strand to which it is bound and preferentially unwinds DNA substrates with a forklike structure.     

Active site specificity of the adenylate cyclase catalytic unit from bovine caudate nucleus

ATP analogues were used to study the active site specificity of the catalytic unit (C) of solubilized and partially purified bovine brain caudate nucleus adenylate cyclase. Phenylenediamine ATP (PD-ATP), 8-azido ATP (8-N3ATP), chromium(III) 3'-beta-alanylarylazido ATP (CrATPa), and 2',3'-dialdehyde ATP (oATP) are competitive inhibitors of C in the presence of the substrate MnATP and the activator forskolin. (Km for MnATP is 50 +/- 11 microM, n = 13). The Ki values determined under initial velocity conditions are: PD-ATP, Ki = 695 +/- 60 microM, n = 5; 8-N3ATP, Ki = 155 +/- 23 microM, n = 5; CrATPa, Ki = 7 +/- 3 microM, n = 2; oATP, Ki = 42 +/- 5 microM, n = 3. Irradiation of 100 microM 8-N3ATP by UV light (254 nm) causes the first-order loss of reagent either in the presence or absence of C. Concomitant irreversible inhibition of C in the presence of 8-N3ATP was more complex and asymptotically approached 50% within 4-6 min. Loss of C activity in controls was 10-20%. The fraction of C covalently modified by 8-N3ATP, alpha, was calculated for each time point of irradiation for an increasing initial concentration ([A]o) of 8-N3ATP. Extrapolated to infinite time of photolysis, the value of alpha reached a final level, termed alpha t whose magnitude depended on [A]o. From these data we calculated an apparent KD of 4.5 microM for 8-N3ATP. ATP protected against the irreversible inhibition due to 8-N3ATP. These data are most consistent with a mechanism of photoaffinity labeling involving equilibrium binding and covalent insertion of 8-N3ATP into the active site. These results indicate that the active site binds analogues of ATP which are considerably modified in the adenine, ribose, and gamma-phosphate portions and that the affinity of C for these analogues is within an order of magnitude of the Km for ATP.