Nonequivalence of the nucleotide binding domains of the ArsA ATPase

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB pump that catalyzes extrusion of the metalloids As(III) and Sb(III), conferring metalloid resistance. The catalytic subunit, ArsA, is an ATPase with two homologous halves, A1 and A2, connected by a short linker. Each half contains a nucleotide binding domain. The overall rate of ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. The results of photolabeling of ArsA with the ATP analogue 8-azidoadenosine 5'-[alpha-(32)P]-triphosphate at 4 degrees C indicate that metalloid stimulation correlates with a >10-fold increase in affinity for nucleotide. To investigate the relative contributions of the two nucleotide binding domains to catalysis, a thrombin site was introduced in the linker. This allowed discrimination between incorporation of labeled nucleotides into the two halves of ArsA. The results indicate that both the A1 and A2 nucleotide binding domains bind and hydrolyze trinucleotide, even in the absence of metalloid. Sb(III) increases the affinity of the A1 nucleotide binding domain to a greater extent than the A2 nucleotide binding domain. The ATP analogue labeled with (32)P at the gamma position was used to measure hydrolysis of trinucleotide at 37 degrees C. Under these catalytic conditions, both nucleotide binding domains hydrolyze ATP, but hydrolysis in A1 is stimulated to a greater degree by Sb(III) than A2. These results suggest that the two homologous halves of the ArsA may be functionally nonequivalent.     

Cys-113 and Cys-422 form a high affinity metalloid binding site in the ArsA ATPase

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB extrusion pump for the trivalent metalloids As(III) and Sb(III). ArsA, the catalytic subunit has two homologous halves, A1 and A2. Each half has a consensus signal transduction domain that physically connects the nucleotide-binding domain to the metalloid-binding domain. The relation between metalloid binding by ArsA and transport through ArsB is unclear. In this study, direct metalloid binding to ArsA was examined. The results show that ArsA binds a single Sb(III) with high affinity only in the presence of Mg(2+)-nucleotide. Mutation of the codons for Cys-113 and Cys-422 eliminated Sb(III) binding to purified ArsA. C113A/C422A ArsA has basal ATPase activity similar to that of the wild type but lacks metalloid-stimulated activity. Accumulation of metalloid was assayed in intact cells, where reduced uptake results from active extrusion by the ArsAB pump. Cells expressing the arsA(C113A/C422A)B genes had an intermediate level of metalloid resistance and accumulation between those expressing only arsB alone and those expressing wild type arsAB genes. The results indicate that, whereas metalloid stimulation of ArsA activity enhances the ability of the pump to reduce the intracellular concentration of metalloid, high affinity binding of metalloid by ArsA is not obligatory for transport or resistance. Yet, in mixed populations of cells bearing either arsAB or arsA(C113A/C422A)B growing in subtoxic concentrations of arsenite, cells bearing wild type arsAB replaced cells with mutant arsA(C113A/C422A)B in less than 1 week, showing that the metalloid binding site confers an evolutionary advantage.     

Characterization of the metalloactivation domain of an arsenite/antimonite resistance pump

The ArsAB extrusion pump encoded by the ars operon of Escherichia coli plasmid R773 confers resistance to the toxic trivalent metalloids arsenite [As(III)] and antimonite [Sb(III)]. The ArsA ATPase, the catalytic subunit of the pump, has two homologous halves, A1 and A2. At the interface of these two halves are two nucleotide-binding domains and a metalloid-binding domain. Cys-113 and Cys-422 have been shown to form a high-affinity metalloid binding site. The crystal structure of ArsA shows two other bound metalloid atoms, one liganded to Cys-172 and His-453, and the other liganded to His-148 and Ser-420. The contribution of those putative metalloid sites was examined. There was little effect of mutagenesis of residues His-148 and Ser-420 on metalloid binding. However, a C172A ArsA mutant and C172A/H453A double mutant exhibited significantly decreased affinity for Sb(III). These results suggest first that there is only a single high-affinity metalloid binding site in ArsA, and second that Cys-172 controls the affinity of this site for metalloid and hence the efficiency of metalloactivation of the ArsAB efflux pump.     

The anion-stimulated ATPase ArsA shows unisite and multisite catalytic activity.

ArsA, an anion-stimulated ATPase, consists of two nucleotide binding domains, A1 in the N terminus and A2 in the C terminus of the protein, connected by a linker. The A1 domain contains a high affinity ATP binding site, whereas the A2 domain has low affinity and it requires the allosteric ligand antimonite for binding ATP. ArsA is known to form a UV-activated adduct with [alpha-(32)P]ATP in the linker region. This study shows that on addition of antimonite, much more adduct is formed. Characterization of the nature of the adduct suggests that it is between ArsA and ADP, instead of ATP, indicating that the adduct formation reflects hydrolysis of ATP. The present study also demonstrates that the A1 domain is capable of carrying out unisite catalysis in the absence of antimonite. On addition of antimonite, multisite catalysis involving both A1 and A2 sites occurs, resulting in a 40-fold increase in ATPase activity. Studies with mutant proteins suggest that the A2 site may be second in the sequence of events, so that its role in catalysis is dependent on a functional A1 site. It is also proposed that ArsA goes through an ATP-bound and an ADP-bound conformation, and the linker region, where ADP binds under both unisite and multisite catalytic conditions, may play an important role in the energy transduction process.     

A kinetic model for the action of a resistance efflux pump

ArsA is the catalytic subunit of the arsenical pump, coupling ATP hydrolysis to the efflux of arsenicals through the ArsB membrane protein. It is a paradigm for understanding the structure-function of the nucleotide binding domains (NBD) of medically important efflux pumps, such as P-glycoprotein, because it has two sequence-related, interacting NBD, for which the structure is known. On the basis of a rigorous analysis of the pre-steady-state kinetics of nucleotide binding and hydrolysis, we propose a model in which ArsA alternates between two mutually exclusive conformations as follows: the ArsA(1) conformation in which the A1 site is closed but the A2 site open; and the ArsA(2) conformation, in which the A1 and A2 sites are open and closed, respectively. Antimonite elicits its effects by sequestering ArsA in the ArsA(1) conformation, which catalyzes rapid ATP hydrolysis at the A2 site to drive ArsA between conformations that have high (nucleotide-bound ArsA) and low affinity (nucleotide-free ArsA) for Sb(III). ArsA potentially utilizes this process to sequester Sb(III) from the medium and eject it into the channel of ArsB.     

Conformational changes in four regions of the Escherichia coli ArsA ATPase link ATP hydrolysis to ion translocation

Structures of ArsA with ATP, AMP-PNP, or ADP.AlF(3) bound at the A2 nucleotide binding site were determined. Binding of different nucleotides modifies the coordination sphere of Mg(2+). In particular, the changes elicited by ADP.AlF(3) provide insights into the mechanism of ATP hydrolysis. In-line attack by water onto the gamma-phosphate of ATP would be followed first by formation of a trigonal intermediate and then by breaking of the scissile bond between the beta- and gamma-phosphates. Motions of amino acid side chains at the A2 nucleotide binding site during ATP binding and hydrolysis propagate at a distance, producing conformational changes in four different regions of the protein corresponding to helices H4-H5, helices H9-H10, helices H13-H15, and to the S1-H2-S2 region. These elements are extensions of, respectively, the Switch I and Switch II regions, the A-loop (a small loop near the nucleotide adenine moiety), and the P-loop. Based on the observed conformational changes, it is proposed that ArsA functions as a reciprocating engine that hydrolyzes 2 mol of ATP per each cycle of ion translocation across the membrane.     

Genetic mapping of the interface between the ArsD metallochaperone and the ArsA ATPase

The ArsD metallochaperone delivers trivalent metalloids, As(III) or Sb(III), to the ArsA ATPase, the catalytic subunit of the ArsAB As(III) efflux pump. Transfer of As(III) increases the affinity of ArsA for As(III), allowing resistance to environmental arsenic concentrations. As(III) transfer is channelled from chaperone to ATPase, implying that ArsD and ArsA form an interface at their metal binding sites. A genetic approach was used to test this hypothesis. Thirteen ArsD mutants exhibiting either weaker or stronger interaction with ArsA were selected by either repressed transactivator yeast two-hybrid or reverse yeast two-hybrid assays. Additionally, Lys-37 and Lys-62 were identified as being involved in ArsD function by site-directed mutagenesis and chemical modification. Substitution at either position with arginine was tolerated, suggesting participation of a positive charge. By yeast two-hybrid analysis K37A and K62A mutants lost interaction with ArsA. All 15 mutations were mapped on the surface of the ArsD structure, and their locations are consistent with a structural model generated by in silico docking. Four are close to metalloid binding site residues Cys-12, Cys-13 and Cys-18, and seven are on the surface of helix 1. These results suggest that the interface involves one surface of helix 1 and the metalloid binding site.     

Caenorhabditis elegans expresses a functional ArsA

Because arsenic is the most prevalent environmental toxin, it is imperative that we understand the mechanisms of metalloid detoxification. In prokaryotes, arsenic detoxification is accomplished by chromosomal and plasmid-borne operon-encoded efflux systems. Bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite (As(III)) and antimonite (Sb(III)). Here, we describe the identification of a Caenorhabditis elegans homolog (asna-1) that encodes the ATPase component of the Escherichia coli As(III) and Sb(III) transporter. We evaluated the responses of wild-type and asna-1-mutant nematodes to various metal ions and found that asna-1-mutant nematodes are more sensitive to As(III) and Sb(III) toxicity than are wild-type animals. These results provide evidence that ASNA-1 is required for C. elegans' defense against As(III) and Sb(III) toxicity. A purified maltose-binding protein (MBP)-ASNA-1 fusion protein was biochemically characterized, and its properties compared with those of ArsAs. The ATPase activity of the ASNA-1 protein was dependent on the presence of As(III) or Sb(III). As(III) stimulated ATPase activity by 2 +/- 0.2-fold, whereas Sb(III) stimulated it by 4.6 +/- 0.15-fold. The results indicate that As(III)- and Sb(III)-stimulated ArsA ATPase activities are not restricted to bacteria, but extend to animals, by demonstrating that the asna-1 gene from the nematode, C. elegans, encodes a functional ArsA ATPase whose activity is stimulated by As(III) and Sb(III) and which is critical for As(III) and Sb(III) tolerance in the intact organism.     

The linker peptide of the ArsA ATPase

Plasmid R773 encodes an As(III)/Sb(III)-translocating ATPase that confers resistance to those metalloids in Escherichia coli. The catalytic subunit of the pump, the ArsA ATPase, consists of homologous N- and C-terminal nucleotide-binding domains connected by a 25-residue linker. The role of this linker sequence was examined by deletion of five, 10, 15 or 23 residues or insertion of five glycine residues. Cells expressing arsA with the 5-residue insertion had wild-type arsenite resistance. Resistance of cells expressing modified arsA genes with deletions was dependent on the linker length. Cells with five or 10 deleted residues exhibited slightly reduced resistance. Deletion of 15 or 23 residues resulted in further decreases in resistance. Each altered ArsA was purified. The enzyme with the 5-residue insertion had the same affinity for ATP and Sb(III) as the wild-type enzyme. Enzymes with 5-, 10-, 15- or 23-residue deletions exhibited decreased affinity for both Sb(III) and ATP. The enzyme with a 23-residue deletion exhibited only basal ATPase activity and was unable to be allosterically activated by Sb(III). These results suggest that the linker has evolved to a length optimal for bringing the two halves of the protein into proper contact with each other, facilitating catalysis.     

Alteration of the nucleotide-binding site asymmetry of chloroplast coupling factor 1 by catalysis

Fluorescence resonance energy transfer was used to show that ATP hydrolysis induces a change in the properties of two nucleotide-binding sites in isolated chloroplast coupling factor 1 (CF1). The fluorescence donor was Lucifer Yellow vinyl sulfone (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide- 3,6-disulfonate), covalently bound to a unique site on the alpha subunit between nucleotide-binding sites 2 and 3. The fluorescence acceptor was the ATP analog 2'(3')-trinitrophenyladenosine 5'-triphosphate (TNP-ATP), incorporated specifically into nucleotide-binding site 1. Energy transfer from Lucifer Yellow to TNP-ATP in site 1 was greater if catalysis occurred before TNP-ATP was incorporated than if no catalysis occurred, indicating that one of the nucleotide-binding sites near the Lucifer Yellow had changed its properties to those of site 1 as a result of catalysis. The amount of energy transfer increased with the degree of substrate excess during catalysis, as expected if catalysis were required for the new site 1 location. ADP, which binds to CF1, but is not a substrate for hydrolysis, caused little energy transfer. Titration of site 3 with TNP-ATP showed greater energy transfer from Lucifer Yellow when catalysis had not occurred, indicating that sites 1 and 3 switched properties as a result of catalysis. The amount of energy transfer declined exponentially with time between removal of substrate and addition of TNP-ATP to site 1, with a half-time of 1.5-2 h at room temperature. This result suggests that the change that results in switching of nucleotide-binding sites 1 and 3 relaxes in the absence of substrate. Our results show that the asymmetry of the nucleotide-binding sites of CF1 is not a permanent feature of the molecule.     

Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding

The ability of subunit C of eukaryotic V-ATPases to bind ADP and ATP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the photoaffinity and the FCS data indicate that the ATP-analogues bind more weakly to subunit C than the ADP-analogues. Site-directed mutagenesis and N-terminal sequencing of subunit C from Arabidopsis (VHA-C) and yeast (Vma5p) have been used to map the C-terminal region of subunit C as the nucleotide-binding site. Tryptophan fluorescence quenching and decreased susceptibility to tryptic digestion of subunit C after binding of different nucleotides provides evidence for structural changes in this subunit caused by nucleotide-binding.     

Role of conserved aspartates in the ArsA ATPase

The ArsA ATPase is the catalytic subunit of the arsenite-translocating ArsAB pump that is responsible for resistance to arsenicals and antimonials in Escherichia coli. ATPase activity is activated by either arsenite or antimonite. ArsA is composed of two homologous halves A1 and A2, each containing a nucleotide binding domain, and a single metalloid binding or activation domain is located at the interface of the two halves of the protein. The metalloid binding domain is connected to the two nucleotide binding domains through two DTAPTGH sequences, one in A1 and the other in A2. The DTAPTGH sequences are proposed to be involved in information communication between the metal and catalytic sites. The roles of Asp142 in A1 D 142TAPTGH sequence, and Asp447 in A2 D 447TAPTGH sequence was investigated after altering the aspartates individually to alanine, asparagine, and glutamate by site-directed mutagenesis. Asp142 mutants were sensitive to As(III) to varying degrees, whereas the Asp447 mutants showed the same resistance phenotype as the wild type. Each altered protein exhibited varying levels of both basal and metalloid-stimulated activity, indicating that neither Asp142 nor Asp447 is essential for catalysis. Biochemical characterization of the altered proteins imply that Asp142 is involved in Mg (2+) binding and also plays a role in signal transduction between the catalytic and activation domains. In contrast, Asp447 is not nearly as critical for Mg (2+) binding as Asp142 but appears to be in communication between the metal and catalytic sites. Taken together, the results indicate that Asp142 and Asp447, located on the A1 and A2 halves of the protein, have different roles in ArsA catalysis, consistent with our proposal that these two halves are functionally nonequivalent.     

The ArsD As(III) metallochaperone

Arsenic, a toxic metalloid widely existing in the environment, causes a variety of health problems. The ars operon encoded by Escherichia coli plasmid R773 has arsD and arsA genes, where ArsA is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump, and ArsD is an arsenic chaperone for ArsA. ArsD transfers As(III) to ArsA and increases the affinity of ArsA for As(III), allowing resistance to environmental concentrations of arsenic. Cys12, Cys13 and Cys18 in ArsD form a three sulfur-coordinated As(III) binding site that is essential for metallochaperone activity. ATP hydrolysis by ArsA is required for transfer of As(III) from ArsD to ArsA, suggesting that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle. The 1.4 Å x-ray crystal structure of ArsD shows a core of four ??-strands flanked by four ??-helices in a thioredoxin fold. Docking of ArsD with ArsA was modeled in silico. Independently ArsD mutants exhibiting either weaker or stronger interaction with ArsA were selected. The locations of the mutations mapped on the surface of ArsD are consistent with the docking model. The results suggest that the interface with ArsA involves one surface of ??1 helix and metalloid binding site of ArsD.     

Aurovertin fluorescence changes of the mitochondrial F1-ATPase during multi- and uni-site ATP hydrolysis

The aurovertin-F1 complex was used to monitor fluorescence changes of the mitochondrial adenosine triphosphatase during multi- and uni-site ATP hydrolysis. It is known that the fluorescence intensity of the complex is partially quenched by addition of ATP or Mg2+ and enhanced by ADP (Chang, T., and Penefsky, H. S. (1973) J. Biol. Chem. 248, 2746-2754). In the present study low concentrations of ATP (0.03 mM) induced a marked fluorescence quenching which was followed by a fast fluorescence recovery. This recovery could be prevented by EDTA or an ATP regenerating system. The rate of ATP hydrolysis by the aurovertin-F1 complex and the reversal of the ATP-induced fluorescence quenching were determined in these various conditions. ITP hydrolysis also resulted in fluorescence quenching that was followed by a recovery of fluorescence intensity. Under conditions for single site catalysis, fluorescence quenching was observed upon the addition of ATP. This strongly indicates that fluorescence changes in the aurovertin-F1 complex are due to the binding and hydrolysis of ATP at a catalytic site. Therefore the resulting ADP molecule bound at this catalytic site possibly induces the fluorescence recovery observed.     

Asp45 is a Mg2+ ligand in the ArsA ATPase.

The ATPase activity of ArsA, the catalytic subunit of the plasmid-encoded, ATP-dependent extrusion pump for arsenicals and antimonials in Escherichia coli, is allosterically activated by arsenite or antimonite. Magnesium is essential for ATPase activity. To examine the role of Asp45, mutants were constructed in which Asp45 was changed to Glu, Asn, or Ala. Cells expressing these mutated arsA genes lost arsenite resistance to varying degrees. Purified D45A and D45N enzymes were inactive. The purified D45E enzyme exhibited approximately 5% of the wild type activity with about a 5-fold decrease in affinity for Mg2+. Intrinsic tryptophan fluorescence was used to probe Mg2+ binding. ArsA containing only Trp159 exhibited fluorescence enhancement upon the addition of MgATP, which was absent in D45N and D45A. As another measure of conformation, limited trypsin digestion was used to estimate the surface accessibility of residues in ArsA. ATP and Sb(III) synergistically protected wild type ArsA from trypsin digestion. Subsequent addition of Mg2+ increased trypsin sensitivity. D45N and D45A remained protected by ATP and Sb(III) but lost the Mg2+ effect. D45E exhibited an intermediate Mg2+ response. These results indicate that Asp45 is a Mg2+-responsive residue, consistent with its function as a Mg2+ ligand.     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＾＋－ＡＴＰ酶复合中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ时,Ｆｏ的荧光猝灭常数的变化结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝来常数,也就是Ｆｏ会产生不同的构象变化。这些结果说明了Ｈ＾＋ＡＴＰ酶合ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＋－ＡＴＰ酶复合体（也称ＡＴＰ合成酶）中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ（酶蛋白与底物分子比为１：１）时,Ｆｏ的荧光猝灭常数的变化（用竹红菌乙作为膜区蛋白荧光的猝灭剂）结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝灭常数（Ｋｓｖ）,也就是Ｆｏ会产生不同的构象变化。加入二价金属离子起动ＡＴＰ水解反应结束后：ＡＴＰ＋Ｈ２Ｏ→ＡＤＰ＋Ｐｉ,这时可以在Ｆｏ观察到与ＡＤＰ加Ｍｇ２＋时相同猝灭常数Ｋｓｖ;用荧光强度随时间进程变化的实验可观察到Ｆ１水解过程中导致Ｆｏ构象变化的动力学过程。这些结果说明了Ｈ＋－ＡＴＰ酶复合体ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

The ATPase mechanism of ArsA, the catalytic subunit of the arsenite pump.

The ArsA ATPase is the catalytic subunit of a novel arsenite pump, with two nucleotide-binding consensus sequences in the N- and C-terminal halves of the protein. The single tryptophan-containing Trp159 ArsA was used to elucidate the elementary steps of the ATPase mechanism by fluorescence stopped-flow experiments. The binding and hydrolysis of MgATP is a multistep process with a minimal kinetic mechanism (Mechanism 1). A notable feature of the reaction is that MgATP binding induces a slow transient increase in fluorescence of ArsA, which is independent of the ATP concentration, indicative of the build-up of a pre-steady state intermediate. This finding, coupled with a phosphate burst, implies that the steady-state intermediate builds up subsequent to product release. We propose that the rate-limiting step is an isomerization between different conformational forms of ArsA. kcat is faster than the phosphate burst, indicating that both nucleotide binding sites of ArsA are catalytic. Consistent with this interpretation, approximately 2 mol of phosphate are released per mole of ArsA during the phosphate burst.     

ATP binding,not hydrolysis,at the first nucleotide-binding domain of multidrug resistance-associated protein MRP1 enhances ADP.Vi trapping at the second domain

Multidrug resistance-associated protein (MRP1) transports solutes in an ATP-dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP. We found that ATP binding to the first NBD of MRP1 increases binding and trapping of ADP at the second domain (Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2002) J. Biol. Chem. 277, 5110-5119). These results were interpreted as indicating that the binding of ATP at NBD1 causes a conformational change in the molecule and increases the affinity for ATP at NBD2. However, we did not distinguish between the possibilities that the enhancement of ADP trapping might be caused by either ATP binding alone or hydrolysis. We now report the following. 1) ATP has a much lesser effect at 0 degrees C than at 37 degrees C. 2) After hexokinase treatment, the nonhydrolyzable ATP analogue, adenyl 5'-(yl iminodiphosphate), does not enhance ADP trapping. 3) Another nonhydrolyzable ATP analogue, adenosine 5'-(beta,gamma-methylene)triphosphate, whether hexokinase-treated or not, causes a slight enhancement. 4) In contrast, the hexokinase-treated poorly hydrolyzable ATP analogue, adenosine 5'-O-(thiotriphosphate) (ATPgammaS), enhances ADP trapping to a similar extent as ATP under conditions in which ATPgammaS should not be hydrolyzed. We conclude that: 1) ATP hydrolysis is not required to enhance ADP trapping by MRP1 protein; 2) with nucleotides having appropriate structure such as ATP or ATPgammaS, binding alone can enhance ADP trapping by MRP1; 3) the stimulatory effect on ADP trapping is greatly diminished when the MRP1 protein is in a "frozen state" (0 degrees C); and 4) the steric structure of the nucleotide gamma-phosphate is crucial in determining whether binding of the nucleotide to NBD1 of MRP1 protein can induce the conformational change that influences nucleotide trapping at NBD2.     

Nucleotide exchange from the high-affinity ATP-binding site in SecA is the rate-limiting step in the ATPase cycle of the soluble enzyme and occurs through a specialized conformational state

We have characterized the kinetic and thermodynamic consequences of adenine nucleotide interaction with the low-affinity and high-affinity nucleotide-binding sites in free SecA. ATP binds to the hydrolytically active high-affinity site approximately 3-fold more slowly than ADP when SecA is in its conformational ground state, suggesting that ATP binding probably occurs when the enzyme is in another conformational state during the productive ATPase/transport cycle. The steady-state ATP hydrolysis rate is equivalent to the rate of ADP release from the high-affinity site under a number of conditions, indicating that this process is the rate-limiting step in the ATPase cycle of the free enzyme. Because efficient protein translocation requires at least a 100-fold acceleration in the ATPase rate, the rate-limiting process of ADP release from the high-affinity site is likely to play a controlling role in the conformational reaction cycle of SecA. This release process involves a large enthalpy of activation, suggesting that it involves a protein conformational change, and two observations indicate that this conformational change is different from the well-characterized endothermic conformational transition believed to gate the binding of SecA to SecYEG. First, nucleotide binding to the low-affinity site strongly inhibits the endothermic transition but does not reduce the rate of ADP release. Second, removal of Mg(2+) from an allosteric binding site on SecA does not perturb the endothermic transition but produces a 10-fold acceleration in the rate of ADP release. These divergent effects suggest that a specialized conformational transition mediates the rate-limiting ADP-release process in SecA. Finally, ADP, 2'-O-(N-methylanthraniloyl)-adenosine-5'-diphosphate (MANT-ADP), and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) bind with similar affinities to the high-affinity site and also to the low-affinity site as inferred from their consistent effects in inhibiting the endothermic transition. In contrast, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) shows 100-fold weaker affinity than ADP for the high-affinity site and no detectable interaction with the low-affinity site at concentrations up to 1 mM, suggesting that this nonhydrolyzable analogue may not be a faithful mimic of ATP in its interactions with SecA.