ATP Cleavage Research Articles

Reverse Gyrase, the Two Domains Intimately Cooperate to Promote Positive Supercoiling

Reverse gyrases are atypical topoisomerases present in hyperthermophiles and are able to positively supercoil a circular DNA. Despite a number of studies, the mechanism by which they perform this peculiar activity is still unclear. Sequence data suggested that reverse gyrases are composed of two putative domains, a helicase-like and a topoisomerase I, usually in a single polypeptide. Based on these predictions, we have separately expressed the putative domains and the full-length polypeptide of Sulfolobus acidocaldarius reverse gyrase as recombinant proteins in Escherichia coli. We show the following. (i) The full-length recombinant enzyme sustains ATP-dependent positive supercoiling as efficiently as the wild type reverse gyrase. (ii) The topoisomerase domain exhibits a DNA relaxation activity by itself, although relatively low. (iii) We failed to detect helicase activity for both the N-terminal domain and the full-length reverse gyrase. (iv) Simple mixing of the two domains reconstitutes positive supercoiling activity at 75 degrees C. The cooperation between the domains seems specific, as the topoisomerase domain cannot be replaced by another thermophilic topoisomerase I, and the helicase-like cannot be replaced by a true helicase. (v) The helicase-like domain is not capable of promoting stoichiometric DNA unwinding by itself; like the supercoiling activity, unwinding requires the cooperation of both domains, either separately expressed or in a single polypeptide. However, unwinding occurs in the absence of ATP and DNA cleavage, indicating a structural effect upon binding to DNA. These results suggest that the N-terminal domain does not directly unwind DNA but acts more likely by driving ATP-dependent conformational changes within the whole enzyme, reminiscent of a protein motor.

New molecular catalysts for ATP cleavage. Criteria of size complementarity

The interaction of the cyclophane receptor 2,5,8,11,14,17-hexaaza[18]metacyclophane (L) with the nucleotides ATP, ADP and AMP is described. L yields one of the largest rate enhancements for hydrolytic ATP cleavage observed in macrocyclic polyamines. The process is specific for the formation of ADP and involves a high degree of geometrical complementarity between host and guest species. The analogue compound 24-hydroxy-2,5,8,11,14,17-hexaaza[18]metacyclophane (L11) shows also a high degree of ATP activation. However, in this case deprotonation of the hydroxy group results in almost complete quenching of the ATPase activity above pH 7.0.

Kinetic analysis of Dictyostelium discoideum myosin motor domains with glycine-to-alanine mutations in the reactive thiol region.

Three conserved glycine residues in the reactive thiol region of Dictyostelium discoideummyosin II were replaced by alanine residues. The resulting mutants G680A, G684A, and G691A were expressed in the soluble myosin head fragment M761-2R [Anson, M., Geeves, M. A., Kurzawa, S. E., and Manstein, D. J. (1996) EMBO J. 15, 6069-6074] and characterized using transient kinetic methods. Mutant G691A showed no major alterations except for a marked increase in basal Mg2+-ATPase activity. Phosphate release seemed to be facilitated by this mutation, and the addition of actin to G691A stimulated ATP turnover not more than 3-fold. In comparison to M761-2R, mutant constructs G691A and G684A showed a 4-fold reduction in the rate of the ATP cleavage step. Most other changes in the kinetic properties of G684A were small ( approximately 2-fold). In contrast, substitution of G680 by an alanine residue led to large changes in nucleotide binding. Compared to M761-2R, rates of nucleotide binding were 20-30-fold slower and the affinity for mantADP was approximately 10-fold increased due to a 200-fold reduction in the dissociation rate constant of mantADP. The ATP-induced dissociation of actin from the acto.680A complex was normal, but the communication between ADP and actin binding was altered such that the two sites are thermodynamically uncoupled but kinetically actin still accelerates ADP release.

Nucleotide Occlusion in the Human Cystic Fibrosis Transmembrane Conductance Regulator: DIFFERENT PATTERNS IN THE TWO NUCLEOTIDE BINDING DOMAINS

The function of the human cystic fibrosis transmembrane conductance regulator (CFTR) protein as a chloride channel or transport regulator involves cellular ATP binding and cleavage. Here we describe that human CFTR expressed in insect (Sf9) cell membranes shows specific, Mg2+-dependent nucleotide occlusion, detected by covalent labeling with 8-azido-[alpha-32P]ATP. Nucleotide occlusion in CFTR requires incubation at 37 degrees C, and the occluded nucleotide can not be removed by repeated washings of the membranes with cold MgATP-containing medium. By using limited tryptic digestion of the labeled CFTR protein we found that the adenine nucleotide occlusion preferentially occurred in the N-terminal nucleotide binding domain (NBD). Addition of the ATPase inhibitor vanadate, which stabilizes an open state of the CFTR chloride channel, produced an increased nucleotide occlusion and resulted in the labeling of both the N-terminal and C-terminal NBDs. Protein modification with N-ethylmaleimide prevented both vanadate-dependent and -independent nucleotide occlusion in CFTR. The pattern of nucleotide occlusion indicates significant differences in the ATP hydrolyzing activities of the two NBDs, which may explain their different roles in the CFTR channel regulation.

Regulation of autophosphorylation of Escherichia coli nitrogen regulator II by the PII signal transduction protein.

The nitrogen regulator II (NRII or NtrB)-NRI (NtrC) two-component signal transduction system regulates the transcription of nitrogen-regulated genes in Escherichia coli. The NRII protein has both kinase and phosphatase activities and catalyzes the phosphorylation and dephosphorylation of NRI, which activates transcription when phosphorylated. The phosphatase activity of NRII is activated by the PII signal transduction protein. We showed that PII was also an inhibitor of the kinase activity of NRII. The data were consistent with the hypothesis that the kinase and phosphatase activities of two-component system kinase/phosphatase proteins are coordinately and reciprocally regulated. The ability of PII to regulate NRII is allosterically controlled by the small-molecule effector 2-ketoglutarate, which binds to PII. We studied the effect of 2-ketoglutarate on the regulation of the kinase and phosphatase activities of NRII by PII, using a coupled enzyme system to measure the rate of cleavage of ATP by NRII. The data were consistent with the following hypothesis: when not complexed with 2-ketoglutarate, PII cannot bind to NRII and has no effect on its competing NRI kinase and phosphatase activities. Under these conditions, the kinase activity of NRII is dominant. At low 2-ketoglutarate concentrations, PII trimers complexed with a single molecule of 2-ketoglutarate interact with NRII to inhibit its kinase activity and activate its phosphatase activity. However, at high 2-ketoglutarate concentrations, PII binds additional ligand molecules and is rendered incapable of binding to NRII, thereby releasing inhibition of NRII's kinase activity and effectively inhibiting its phosphatase activity (by failing to stimulate it).

Kinetic parameters and tissue distribution of 5-oxo- l-prolinase determined by a fluorimetric assay

5-Oxo- l-prolinase (5-OPase) catalyses the hydrolysis of 5-oxo- l-proline to glutamate with concomitant stoichiometric cleavage of ATP to ADP, a reaction which is known to be part of the γ-glutamyl cycle—an interrelated series of reactions involved in the synthesis and metabolism of glutathione. As recent studies indicate, this cyclic pathway plays a crucial role in the regulation of amino acid transport. Apparently, the intermediate product 5-oxo- l-proline functions as a second messenger molecule that upregulates the activity of certain amino acid transport systems. Thus, the degradation of 5-oxo- l-proline by 5-OPase leads to the downregulation of this stimulus. In this study, a new sensitive fluorimetric assay for 5-OPase activity was established which is based on the derivatization of glutamate with o-phthaldialdehyde in the presence of thiols and subsequent separation of the products by HPLC. The method is suitable for the screening of chromatography fractions as well as for the determination of the kinetic parameters K m and V max of purified 5-OPase. Additionally, it can be used for the measurement of enzyme activity in crude cell extracts and evaluation of tissue distribution.

Phosphorylations of αA- and αB-crystallin

In addition to being refractive proteins in the vertebrate lens, the two α-crystallin polypeptides ( αA and αB) are also molecular chaperones that can protect proteins from thermal aggregation. The αB-crystallin polypeptide, a functional member of the small heat shock family, is expressed in many tissues in a developmentally regulated fashion, is stress-inducible, and is overexpressed in many degenerative diseases and some tumors indicating that it plays multiple roles. One possible clue to α-crystallin functions is the fact that both polypeptides are phosphorylated on serine residues by cAMP-dependent and cAMP-independent mechanisms. The cAMP-independent pathway is an autophosphorylation that has been demonstrated in vitro, depends on magnesium and requires cleavage of ATP. Disaggregation of αA-, but not αB-crystallin into tetramers results in an appreciable increase in autophosphorylation activity, reminiscent of other heat shock proteins, and suggests the possibility that changes in the aggregation state of αA-crystallin are involved in yet undiscovered signal transduction pathways. The α-crystallin polypeptides differ with respect to their abilities to undergo cAMP-dependent phosphorylation, with preference given to the αB-crystallin chain. These differences and complexities in α-crystallin phosphorylations, coupled with the differences in expression patterns of the two α-crystallin polypeptides, are consistent with the idea that each polypeptide has distinctive structural and metabolic roles.

Molecular motors: The natural economy of kinesin

Kinesin is a molecular-scale walking machine. New analyses of its mechanism indicate that each step along a microtubule consumes one ATP molecule, and that the binding and cleavage of ATP precede detachment of the molecule's ‘feet’. Directional walking ensues if ATP processing occurs preferentially on one foot.

Coupling of protein surface hydrophobicity change to ATP hydrolysis by myosin motor domain

Dielectric spectroscopy with microwaves in the frequency range between 0.2 and 20 GHz was used to study the hydration of myosin subfragment 1 (S1). The data were analyzed by a method recently devised, which can resolve the total amount of water restrained by proteins into two components, one with a rotational relaxation frequency (fc) in the gigahertz region (weakly restrained water) and the other with lower fc (strongly restrained water). The weight ratio of total restrained water to S1 protein thus obtained (0.35), equivalent to 2100 water molecules per S1 molecule, is not much different from the values (0.3-0.4) for other proteins. The weakly restrained component accounts for about two-thirds of the total restrained water, which is in accord with the number of water molecules estimated from the solvent-accessible surface area of alkyl groups on the surface of the atomic model of S1. The number of strongly restrained water molecules coincides with the number of solvent-accessible charged or polar atoms. The dynamic behavior of the S1-restrained water during the ATP hydrolysis was also examined in a time-resolved mode. The result indicates that when S1 changes from the S1.ADP state into the S1.ADP.P1 state (ADP release followed by ATP binding and cleavage), about 9% of the weakly restrained waters are released, which are restrained again on slow P1 release. By contrast, there is no net mobilization of strongly restrained component. The observed changes in S1 hydration are quantitatively consistent with the accompanying large entropy and heat capacity changes estimated by calorimetry (Kodama, 1985), indicating that the protein surface hydrophobicity change plays a crucial role in the enthalpy-entropy compensation effects observed in the steps of S1 ATP hydrolysis.

Poly-L-lysine Activates both Peptide and ATP Hydrolysis by the ATP-Dependent HslVU Protease inEscherichia coli

HslVU inE. coliis a new type of ATP-dependent protease composed of two heat shock proteins, the HslU ATPase and the HslV peptidase related to certain β-type subunits of the 20S proteasome. Here we show that the ATP-dependent hydrolysis ofN-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methylcoumarin by the HslVU protease can be markedly stimulated by poly-L-lysine, that is known to activate the casein-degrading activity of the 20S proteasome. However, poly-L-lysine showed little or no effect on the peptidase activity of HslV itself. Instead, it stimulated the hydrolysis of ATP by HslU several-fold. Histone that could stimulate the ATPase activity of HslU also increased the rate of the ATP-dependent peptide hydrolysis by HslV, although to a much lesser extent than by poly-L-lysine. Thus, the poly-L-lysine-mediated increase in the ATPase activity of HslU appears to be responsible for the dramatic activation of the ATP-dependent peptide hydrolysis by HslV. These results suggest that, in the reconstituted HslVU complex, the peptide hydrolysis by HslV occurs in a tightly coupled process with the cleavage of ATP by HslU.

Purification and Characterization of the Heat Shock Proteins HslV and HslU That Form a New ATP-dependent Protease in Escherichia coli

The hslVU operon in Escherichia coli encodes two heat shock proteins, HslV, a 19-kDa protein homologous to beta-type subunits of the 20 S proteasomes, and HslU, a 50-kDa protein related to the ATPase ClpX. We have recently shown that HslV and HslU can function together as a novel ATP-dependent protease, the HslVU protease. We have now purified both proteins to apparent homogeneity from extracts of E. coli carrying the hslVU operon on a multicopy plasmid. HslU by itself cleaved ATP, and pure HslV is a weak peptidase degrading certain hydrophobic peptides. HslU dramatically stimulated peptide hydrolysis by HslV when ATP is present. With a 1:4 molar ratio of HslV to HslU, approximately a 200-fold increase in peptide hydrolysis was observed. HslV stimulated the ATPase activity of HslU 2-4-fold, but had little influence on the affinity of HslU to ATP. The nonhydrolyzable ATP analog, beta,gamma-methylene-ATP, did not support peptide hydrolysis. Other nucleotides (CTP, dATP) that were slowly hydrolyzed by HslU allowed some peptide hydrolysis. Therefore, ATP cleavage appears essential for the HslV activity. Upon gel filtration on a Sephacryl S-300 column, HslV behaved as a 250-kDa oligomer (i.e. 12-14 subunits), and HslU behaved as a 100-kDa protein (i.e. a dimer) in the absence of ATP, but as a 450-kDa multimer (8-10 subunits) in its presence. Therefore ATP appears necessary for oligomerization of HslU. Thus the HslVU protease appears to be a two-component protease in which HslV harbors the peptidase activity, while HslU provides an essential ATPase activity.

Effects of acetyl CoA on the pre-steady-state kinetics of the biotin carboxylation reaction of pyruvate carboxylase.

The approach to steady-state for the formation of the enzyme-carboxybiotin complex obeys first-order kinetics, with the proportion of the total enzyme present as the enzyme-carboxybiotin complex in the steady-state being about 60%. The approach to steady-state for ATP cleavage also obeys first-order kinetics. The apparent first-order rate constants for the approach to steady-state, in the presence and absence of acetyl CoA, respectively, are 6.6 and 0.028 s(-1) for ATP cleavage and 6.1 and 0.028 s(-1) for enzyme-carboxybiotin formation. The similarities of the values of the rate constants for the two reactions indicates that there is a common rate-limiting step. The large enhancement of these rate constants in the presence of acetyl CoA suggests that a major effect of acetyl CoA in the reaction is to enhance the rate of the step in which the putative carboxyphosphate complex is formed and in which ATP is cleaved. In addition, in the presence of acetyl CoA, the formation of the enzyme-carboxybiotin complex is much more tightly coupled to ATP cleavage in the presence of acetyl CoA than in its absence. Modeling studies were performed, and reaction schemes are proposed which give simulations similar to the experimental data. In the reaction schemes, the carboxyphosphate intermediate is able to undergo abortive decomposition without carboxylating biotin. The rate of this abortive reaction is greatly reduced in the presence of acetyl CoA.

Determination of the chemical pathway for 4-chlorobenzoate:coenzyme A ligase catalysis.

4-Chlorobenzoate:coenzyme A ligase (4-CBA:CoA ligase) catalyzes the first step of the 4-CBA degradation pathway of Pseudomonas sp. strain CBS3. In this reaction, 4-CBA-CoA thioester synthesis is coupled to ATP cleavage. The studies described in this paper examine the intermediacy of 4-chlorobenzoyl-adenosine 5'phosphate diester (4-CBA-AMP) in the ligase reaction. The 4-CBA-AMP adduct was isolated from the ligase reaction mixture generated from magnesium adenosine 5-triphosphate (MgATP) and 4-CBA in the absence of CoA. The structure of the 4-CBA-AMP was verified by 1H- 13C-, and 31P-nuclear magnetic resonance analysis. Single-turnover reactions carried out with 14C-labeled 4-CBA in a rapid quench apparatus demonstrated formation of the enzyme. 4-CBA-AMP.MgPPi complex from the enzyme.4-CBA.MgATP complex at a rate of 135 s-1. The rate of ligand release from the enzyme.4-CBA-AMP.MgPPi complex was measured at 0.013 s-1. Single-turnover reactions of [14C]-4-CBA, MgATP, and CoA catalyzed by the ligase revealed that the 4-CBA-AMP intermediate formed reaches a maximum level of 25% of the starting 4-CBA within 10 ms and then declines with the formation of the 4-CBA-CoA. The rates of the adenylation and thioesterification partial reactions, determined by kinetic simulation of the rate data, are nearly equal (135 and 100 s-1). Substitution of CoA with the slow substrate pantetheine did not significantly alter the rate of the adenylation step but did reduce the rate of the thioesterification step to 2 s-1. The maximum level of 4-CBA-AMP reached during the single-turnover reaction of 4-CBA, MgATP, and pantetheine corresponded to one-half of the starting 4-CBA.

Effector-mediated stimulation of ATPase activity by the sigma 54-dependent transcriptional activator FHLA from Escherichia coli

The FHLA protein is the transcriptional regulator of the genes of the formate regulon from Escherichia coli. The protein shares homology with the sigma 54-dependent regulators of the NTRC family in the central and C-terminal domains but differs in possessing an extended N terminus lacking the aspartate residue which is the site of phosphorylation. Purified FHLA displays intrinsic ATPase activity which is stimulated weakly by formate and DNA. The presence of both formate and DNA carrying the upstream regulatory sequence to which FHLA binds leads to a large increase in the rate of ATP hydrolysis. Hypophosphite, a structural analog of formate, and azide, a transition state analog of formate, also stimulate ATPase activity, supporting the conclusion that formate is a direct ligand of FHLA. Half-maximal saturation of FHLA with formate took place at around 5 mM, and half-maximal saturation with target DNA took place at around 50 nM. The stimulation of ATPase activity by formate was conferred by a decrease in the apparent Km for ATP, whereas the effect of the DNA binding site also affected the Kcat of the reaction. The other nucleoside triphosphates, GTP, UTP, and CTP, competed with ATP cleavage by FHLA, suggesting at least their binding to FHLA. The specific ATPase activity of FHLA was dependent on the concentration of FHLA in the assay, especially in the presence of DNA and formate. Direct liganding of the effector, therefore, leads to the same consequence as phosphorylation for the NTRC-type regulators, namely, stimulation of ATPase activity.

A structural and kinetic study on myofibrils prevented from shortening by chemical cross-linking.

In previous work, we studied the early steps of the Mg(2+)-ATPase activity of Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. The myofibrils were free to contract, and the results obtained refer to the ATPase cycle of myofibrils contracting with no external load. Here we studied the ATPase of myofibrils contracting isometrically. To prevent shortening, we cross-linked them with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). SDS-PAGE and Western blot analyses showed that the myosin rods were extensively cross-linked and that 8% of the myosin heads were cross-linked to the thin filament. The transient kinetics of the cross-linked myofibrils were studied in 0.1 M potassium acetate, pH 7.4 and 4 degrees C, by the rapid-flow quench method. The ATP binding steps were studied by the cold ATP chase and the cleavage and release of products steps by the Pi burst method. In Pi burst experiments, the sizes of the bursts were equal within experimental error to the ATPase site concentrations (as determined by the cold ATP chase methods) for both cross-linked (isometric) and un-cross-linked (isotonic) myofibrils. This shows that in both cases the rate-limiting step is after the cleavage of ATP. When cross-linked, the kcat of Ca(2+)-activated myofibrils was reduced from 1.7 to 0.8 s-1. This is consistent with the observation that fibers shortening at moderate velocity have a higher ATPase activity than isometric fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Bicarbonate-dependent ATP cleavage catalysed by pyruvate carboxylase in the absence of pyruvate.

Preparations of pyruvate carboxylase catalyse the cleavage of MgATP in the absence of pyruvate and acetyl-CoA. The rate of this cleavage is higher in the presence of HCO3- than in its absence. Incubation of the enzyme preparations with an excess of the pyruvate carboxylase inhibitor, avidin, completely abolishes the pyruvate carboxylating activity of the enzyme preparations but only abolishes the HCO3(-)-dependent MgATP cleaving activity, with no effect on the HCO3(-)-independent ATPase activity. The HCO3(-)-dependent MgATP cleavage is also sensitive to inhibition by a pyruvate carboxylase inhibitor, oxamate, and the dependence of the reaction on the free Mg2+ concentration is similar to that of the pyruvate-carboxylation reaction, whereas the HCO3(-)-independent MgATP cleavage is not dependent on the concentration of free Mg2+ in the range tested. This indicates that MgATP cleavage by pyruvate carboxylase is entirely dependent on the presence of HCO3- and that there may be a low level of ATPase contamination in the enzyme preparations. In addition, inhibition of the HCO3(-)-dependent MgATP cleavage by both avidin and oxamate indicate that although biotin does not directly participate in the reaction, its presence is required in that part of the active site of the enzyme. The rate of HCO3(-)-dependent MgATP cleavage is about 0.07% of that of the full pyruvate carboxylation reaction under similar conditions with saturating substrates. The reaction mechanism is sequential with respect to MgATP and HCO3- addition and Mg2+ adds at equilibrium before MgATP. Acetyl-CoA stimulates the HCO3(-)-dependent MgATP cleavage at low MgATP concentrations, with the stimulation being greater at low Mg2+ concentrations. At high levels of MgATP in the presence of acetyl-CoA, substrate inhibition is evident and is more pronounced at increasing concentrations of Mg2+. This inhibition appears to be, at least in part, caused by inhibition of decarboxylation of the enzyme-carboxybiotin complex by the binding to this complex of Mg2+ and MgATP, which probably act to reduce the rate of movement of carboxybiotin from the site of the MgATP cleavage reaction to that of the pyruvate carboxylation reaction where it is unstable and decarboxylates.

Isolation of a carboxyphosphate intermediate and the locus of acetyl-CoA action in the pyruvate carboxylase reaction.

When chicken liver pyruvate carboxylase was incubated with either H14CO3- or gamma-[32P]ATP, a labeled carboxyphospho-enzyme intermediate could be isolated. The complex was catalytically competent, as determined by its subsequent ability to transfer either 14CO2 to pyruvate or 32P to ADP. While the carboxyphospho-enzyme complex was inherently unstable and the stoichiometry of the transfer was variable depending on experimental conditions, both the [14C]carboxyphospho-enzyme and the carboxy[32P]phospho-enzyme had similar half-lives. Acetyl-CoA was shown to be involved in the conversion of the carboxyphospho-enzyme complex to the more stable carboxybiotin-enzyme species, which was consistent with the effects of acetyl-CoA on isotope exchange reactions involving ATP. We were unable to detect the formation of a phosphorylated biotin derivative during the ATP cleavage reaction. In the presence of K+ and at pH 9.5, the acetyl-CoA-independent activity of chicken liver pyruvate carboxylase approached 2% of the acetyl-CoA-stimulated rate, which represents a 30-fold increase on previously reported activity for this enzyme.

Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin-ligated proteins by closely linked mechanisms.

It is known that two types of high-molecular-mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP-dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney. In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 x g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular-sieve chromatography on a Biogel A-1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density-gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21-31 kDa and 13-15 protein components ranging in molecular mass over 35-110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I-labeled lysozyme-ubiquitin conjugates in an ATP-dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP-dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy-dependent, ubiquitin-mediated protein degradation.

Selenoprotein synthesis in E. coli

The product of the selD gene from Escherichia coli catalyses the formation of an activated selenium compound which is required for the synthesis of Sec-tRNA (Sec, selenocysteine) from Ser-tRNA and for the formation of the unusual nucleoside 5-methylaminomethyl-2-selenouridine in several tRNA species. selD was overexpressed in a T7 promoter/polymerase system and purified to apparent homogeneity. Purified SELD protein is a monomer of 37 kDa in its native state and catalyses a selenium-dependent ATP-cleavage reaction delivering AMP and releasing the beta-phosphate as orthophosphate. The gamma-phosphate group of ATP was not liberated in a form able to form a complex with molybdate. It was precluded that any putative covalent or non-covalent ligand of SELD not removed during purification participated in the reaction. In a double-labelling experiment employing [75Se]selenite plus dithiothreitol and [gamma-32P]ATP the 75Se and 32P radioactivities co-chromatographed on a poly(ethyleneimine)-cellulose column. No radioactivity originating from ATP eluted in this position when [alpha-32P]ATP or [beta-32P]ATP or [14C]ATP were offered as substrates. The results support the speculation that the product of SELD is a phosphoselenoate with the phosphate moiety derived phosphoselenoate from the gamma-phosphate group of ATP. The alpha,beta cleavage of ATP is also supported by the finding that neither adenosine 5'-[alpha,beta-methylene]triphosphate nor adenosine 5'-[beta,gamma-methylene]triphosphate served as substrates in the reaction.

Critical analysis of objections against the biological molecular energy machines.

In the past, two important objections against McClare's idea of biological molecular energy machines were raised. One of the criticisms was concerned with the origin of energy gained in ATP cleavage and with an interpretation of McClare's "excited vibrational state." The former argument reveals a failure of the critics to comprehend McClare's approach. As to the excited vibrational state, it can be identified with nonequilibrium conformational states of the unit rather than with a single vibrational mode. The other criticism based on Brillouin's energy cost of measurement argued that reversible operation of biological molecular energy machines would be virtually impossible. Using propagation velocities of deformations of the unit's structure (instead of velocity of light), the objections against reversibility are invalidated even in the framework of the critic's approach. McClare's idea and relevant definitions are thus physically correct.