Thermodynamic constraints on kinetic proofreading in biosynthetic pathways.

We develop a quantitative theory of kinetic proofreading with an arbitrary number of checking steps after the hydrolysis of a nucleoside triphosphate. In particular, we investigate the relationship between the minimum dissipation of free energy required for a given error frequency in such systems. Several conclusions can be drawn from the present treatment: first, the ultimate accuracy of error correcting selective pathways is set by the displacement from equilibrium of the nucleoside triphosphates. Second, it is advantageous to achieve a desired accuracy at a small energy dissipation with several checking steps rather than a single one. This could explain antinomies in the amino acylation reaction as well as in mRNA translation, where small structural differences lead to large differences in flow rates between right and wrong substrates. Third, all checking steps should contribute equally to the accuracy, which implies a specific and symmetrical set of rate constants for the checking events on the enzyme.     

Accuracy of proofreading with zero energy cost

Accuracy of biological discrimination at the molecular level is known in some systems to involve kinetic proofreading mechanisms. Hopfield and Ninio were the first to propose simple specific kinetic mechanisms for such proofreading and to demonstrate that an energy cost accompanies their improvement in accuracy. Savageau and Freter subsequently derived the explicit cost-accuracy relationship for a broad class of proofreading mechanisms, including the conventional Hopfield-Ninio mechanism just referred to. In other systems, the presence of proofreading mechanisms is in question because the diagnostic features of conventional kinetic proofreading are absent. However, Hopfield has recently proposed an alternative “energy-relay” mechanism, which lacks the characteristic features of conventional proofreading and yet is capable of improving accuracy. In this paper, I use the general cost-accuracy relationship that we have previously derived to examine the energy cost and accuracy of proofreading mechanisms involving an energy relay. The principal findings are the following. First, such mechanisms improve accuracy with a zero cost of proofreading, when “proofreading cost,” defined as the cost due specifically to proofreading, is separated from the costs of putting material through the system. Second, the basic energy-relay mechanism discussed by Hopfield has only a modest improvement in accuracy, but a comparable improvement by a conventional proofreading mechanism would have a cost of about 0·0352 (moles ATP per mole of total product output). Third, accuracy can be increased somewhat if multiple stages of conventional kinetic proofreading precede the energy-relay mechanism. The cost for this improvement is zero while a comparable increase in accuracy achieved by conventional proofreading alone has a cost of about 0·0385. Finally, I propose an alternative arrangement of energy-relay mechanisms that is capable of increasing accuracy still further. The maximum accuracy achieved by this scheme at zero energy cost is comparable to that achieved by an infinite expenditure of energy in a single stage of conventional proofreading.     

Acid nucleoside triphosphatase: partial purification and characterization of a new enzyme from human serum

Acid nucleoside triphosphatase (Acid NTPase), an enzyme which catalyzes the hydrolysis of all nucleoside triphosphates to the corresponding diphosphates was purified from human serum with a purification factor of 190 and a recovery of 31%. The molecular weight was 75,000 as estimated by gel filtration. Gel-electrophoresis revealed an Rf-value of 0.11, and the isoelectric point was determined at pH 4.4. It exhibited a temperature optimum of 44 degrees C and the activation energy was estimated to be 41.6 kJ/mol. The enzyme was active in the absence of divalent cations, since activity was not inhibited by EDTA. The presence of this chelator reduced the Km-value from 70 to 40 microM. Inhibitor experiments revealed that tartrate was a weak mixed-type noncompetitive inhibitor, Ki = 88 mM. The enzyme was specific for the hydrolysis of nucleoside triphosphates. P-nitrophenyl phosphate was not accepted as a substrate. The enzyme revealed optimum activity at the exceptionally acid pH of 3.0. These unique characteristics indicate the presence of a novel enzyme.     

Stress, order and survival

Much of the sophisticated chemistry of life is accomplished by multicomponent complexes, which act as molecular machines. Intrinsic to their accuracy and efficiency is the energy that is supplied by hydrolysis of nucleoside triphosphates. Conditions that deplete energy sources should therefore cause decay and death. But studies on organisms that are exposed to prolonged stress indicate that this fate could be circumvented through the formation of highly ordered intracellular assemblies. In these thermodynamically stable structures, vital components are protected by a physical sequestration that is independent of energy consumption.     

Proofreading systems of multiple stages for improved accuracy of biological discrimination

The phenomenal accuracy of biological discrimination is due in many cases to specific proofreading mechanisms. We have previously developed a macroscopic theory of such mechanisms and applied it to the case of single-stage proofreading. In this article we apply the theory to systems with multiple stages of proofreading. A specific relationship between improved accuracy due to proofreading and the associated energy cost is given. This is a macroscopic relationship that must be satisfied regardless of the details of the underlying mechanisms. Five factors in the design of such systems are shown to influence their overall accuracy: (1) initial discrimination, (2) number of proofreading stages, (3) proofreading discrimination of each stage, (4) distribution of proofreading effort among the stages, and (5) total energy expended for proofreading. We show that there is an optimal distribution of proofreading effort that, for a given degree of accuracy, minimizes the energy cost of proofreading. We also provide a simple physical interpretation of this minimum condition. These results are used to examine proofreading in two experimental systems for which there is appropriate data available in the literature: the valyl-tRNA synthetase catalyzed misacylation of tRNA^Val with threonine and the isoleucyl-tRNA synthetase catalyzed misacylation of tRNA^Ile with valine. The correlation between the magnitude of a discrimination factor and the size of the corresponding enzymatic cavity is discussed.     

Increased substrate selectivity during transition from Ca2+-activated to K+,EDTA-activated nucleoside triphosphatase activity of heavy meromyosin

A comparison of kinetic parameters (Km(app) and V) of hydrolysis by heavy meromyosin of natural (ATP and ITP) and modified nucleoside triphosphates showed that in the K+, EDTA-ATPase conformation the enzyme exhibited a higher selectivity towards the structure of the substrate nucleoside moiety than in the case of the Ca2+-stimulated nucleoside triphosphatase activity. In the presence of Ca2+, all the N1- and N6-substituted analogs of ATP as well as ITP, etheno-ATP and the dialdehyde derivative of ATP were hydrolyzed at a high rate irrespective of their markedly decreased affinity for heavy meromyosin. In the presence of K+, EDTA the ATPase activity showed a tendency for a total decrease of the analog affinity for nucleoside triphosphates, i.e., the impossibility of tight binding of the substrate phosphate residues to the protein in the absence of bivalent cations, which was concomitant with an increase in the hydrolysis rate. However, it was found that only in N1-substituted analogs any appreciable changes in the substrate properties were absent. All the other nucleoside triphosphates tested (N6-carboxy-methoxy-ATP, N6-(N'-acetylaminoethoxy)-ATP, etheno-ATP, ITP and the dialdehyde derivative of ATP having a rupture in the ribose ring) lost their ability to be hydrolyzed by heavy meromyosin. The experimental results as well as the literature data are suggestive of differences in the spatial structure of the active center in two different myosin conformations associated with a high catalytic activity, i.e., K+, EDTA-ATPase and Ca2+-ATPase.     

Kinetics of DNA renaturation catalyzed by the RecA protein of Escherichia coli

The recA enzyme of Escherichia coli catalyzes renaturation of DNA coupled to hydrolysis of ATP. The rate of enzymatic renaturation is linearly dependent on recA protein concentration and shows saturation kinetics with respect to DNA concentration. The kinetic analysis of the reaction indicates that the Km for DNA is 65 microM while the kcat is approximately 48 pmol of duplex formed (pmol of recA)-1 (20 min)-1. RecA protein catalyzed renaturation has been characterized with respect to salt sensitivity, Mg2+ ion and pH optima, requirements for nucleoside triphosphates, and inhibition by nonhydrolyzable nucleoside triphosphates and analogues. These results are consistent with a Michaelis-Menten mechanism for DNA renaturation catalyzed by recA protein. A model is described in which oligomers of recA protein bind rapidly to single-stranded DNA, and in the presence of ATP, these nucleoprotein intermediates aggregate to bring complementary sequences into close proximity for homologous pairing. As with other DNA pairing reactions catalyzed by recA protein, ongoing DNA hydrolysis is required for renaturation. However, unlike the strand assimilation or transfer reaction, renaturation is inhibited by E. coli helix-destabilizing protein.     

Characterization of ecto-nucleoside triphosphatase on A-431 human epidermoidal carcinoma cells.

Hydrolysis of extracellular ATP and other nucleoside phosphates by A-431 human epidermoidal carcinoma cells was studied. The hydrolysis of extracellular ATP by these cells required either Mg2+ or Ca2+, and either cation could be replaced by Co2+, Fe2+, or Mn2+. Nucleoside triphosphates (ATP, GTP, CTP, UTP, and dTTP), but not nucleoside diphosphates, were hydrolyzed by the cells with Km and Vmax values similar to those for ATP (0.9-1.1 mmol/l and 6-10 nmol Pi formed/10(6) cells, respectively). The hydrolysis of ATP was inhibited strongly by ATP-gamma S and AMPPNP, and weakly by AMPCPP and ADP-beta S, but not by AMPCPP or AMPCP. Since the hydrolysis of [gamma-32P]ATP was inhibited by all these nucleoside triphosphates, the binding site for ATP is presumed to be the same as that for the other nucleoside triphosphates. All these results indicate that ecto-ATPase activity associated with A-431 cells is due to ecto-nucleoside triphosphatase. The nucleotide specificity shown in the present study indicates that ecto-nucleoside triphosphatase associated with A-431 cells is a molecule different from P2-purinergic receptors which can be stimulated specifically with nucleoside phosphates like ATP, ADP, UTP, UDP, and GTP, but not by other nucleotides.     

Characterization of the mutT nucleoside triphosphatase of Escherichia coli.

The mutT protein, which prevents A:T----C:G transversions during DNA replication, has the following enzymatic properties. Although it prefers dGTP as a substrate, it hydrolyzes all of the canonical nucleoside triphosphates to some extent. It has no activity in the absence of divalent cations, is maximally activated by magnesium ions, and has a pH optimum of 9.0. Nucleoside triphosphates are hydrolyzed according to the following equation. dGTP----dGMP + PPi Studies with nucleotide analogues suggest that the enzyme may prefer the syn rather than the anti conformation of the nucleoside triphosphates, which might explain the role it plays in preventing mutations.     

Effect of nucleotides and inhibitory anions on mitochondrial ATPase. Its pH dependence

The effect of pH on the sensitivity of F1-ATPase as well as mitochondrial ATPase activity to nucleoside diand triphosphates and to inhibitory anions such as cyanate and thiocyanate, has been studied. The results obtained show that nucleotides could act as activators or inhibitors of the ATPase hydrolytic activity depending on pH, substrate concentration, and binding of the enzyme to the membrane. The effect of those nucleotides which activate the hydrolysis of ATP-Mg2+ was more pronounced beyon the optimum pH corresponding to each of the three catalytic sites of the enzyme, whereas those which are inhibitors had a lower effect above this value. The sensitivity to the inhibitory anions decreased with increasing pH values; the decrease in the inhibitory effect was sharper when approaching the optimum pH value. These data are in agreement with the existence in mitochondrial ATPase of two different regulatory sites, one being specific for binding nucleotides, and another for anions. Both of them showed a different response upon changes of pH.     

A new twist on RNA helicases: DExH/D box proteins as RNPases

DExH/D box proteins are required for the major transactions of RNA, including mRNA synthesis, pre-mRNA splicing, ribosome biogenesis, translation and RNA decay. In the popular imagination, DExH/D box proteins have become synonymous with 'RNA helicases', which are enzymes that unwind duplex RNAs in concert with the hydrolysis of nucleoside triphosphates (NTPs). But all DExH/D box proteins may not be RNA helicases and the energy of NTP hydrolysis by DExH/D box proteins may be harnessed for other purposes. Cellular RNAs are associated with proteins, often in large ribonucleoprotein (RNP) complexes. This review focuses on recent progress suggesting a role for DExH/D box proteins as 'RNPases' that use chemical energy to remodel the interactions of RNA and proteins.     

Correlation of an adenine-specific conformational change with the ATP-dependent peptidase activity of Escherichia coli Lon

Escherichia coli Lon, also known as protease La, is a serine protease that is activated by ATP and other purine or pyrimidine triphosphates. In this study, we examined the catalytic efficiency of peptide cleavage as well as intrinsic and peptide-stimulated nucleotide hydrolysis in the presence of hydrolyzable nucleoside triphosphates ATP, CTP, UTP, and GTP. We observed that the k(cat) of peptide cleavage decreases with the reduction in the nucleotide binding affinity of Lon in the following order: ATP > CTP > GTP approximately UTP. Compared to those of the other hydrolyzable nucleotide triphosphates, the ATPase activity of Lon is also the most sensitive to peptide stimulation. Collectively, our kinetic as well as tryptic digestion data suggest that both nucleotide binding and hydrolysis contribute to the peptidase turnover of Lon. The kinetic data that were obtained were further put into the context of the structural organization of Lon protease by probing the conformational change in Lon bound to the different nucleotides. Both adenine-containing nucleotides and CTP protect a 67 kDa fragment of Lon from tryptic digestion. Since this 67 kDa fragment contains the ATP binding pocket (also known as the alpha/beta domain), the substrate sensor and discriminatory (SSD) domain (also known as the alpha-helical domain), and the protease domain of Lon, we propose that the binding of ATP induces a conformational change in Lon that facilitates the coupling of nucleotide hydrolysis with peptide substrate delivery to the peptidase active site.     

Adrenal cortex mitochondrial enzyme with ATP-dependent protease and protein-dependent ATPase activities. Purification and properties

We have purified an ATP-dependent protease with protein-dependent ATPase activity from bovine adrenal cortex mitochondria to near homogeneity. The subunit molecular weight is 108,000 and the enzyme appears to be a hexamer with approximately identical subunits. Based on the experiments using various nucleoside triphosphates and their related compounds, it is concluded that hydrolysis of the high-energy bond in nucleoside triphosphates is not an absolute requirement for proteolysis. Nucleotide specificity of this enzyme varies, depending on the protein or peptide substrates used. When casein was the substrate, ATP and dATP were quite effective, but other nucleotides were not. When insulin and angiotensinogen were used as substrate, ATP, other nucleoside triphosphates, ADP, inorganic triphosphate, pyrophosphate, and phosphate were effective. One of the cleaving linkages hydrolyzed by this enzyme was revealed to be the Leu-Leu bond of angiotensinogen. However, the specificity appears to be broad in view of the hydrolysis pattern of glucagon.     

Kinetic properties of a nucleoside phosphotransferase of chick embryo

1. A nonspecific nucleoside phosphotransferase (nucleotide : 3'-deoxynucleotide 5'-phosphotransferase, EC 2.7.1.77), purified from chick embryos, catalyzes the transfer of phosphate ester from a nucleotide donor to a nucleoside acceptor. 2. The enzyme exhibits sigmoidal kinetics with respect to nucleoside monophosphate donors, but with respect to nucleoside di- or triphosphate donors and nucleoside acceptors hyperbolic kinetics were obtained. 3. The nucleoside phosphotransferase of chick embryo is unstable to heat and is protected from inactivation by a large number of nucleosides. 4. Nucleoside di- and triphosphates lower both the concentration of nucleoside monophosphates required for half-maximal velocity and the kinetic order of reaction measured with these phosphate donors. On the contrary, nucleoside di- or triphosphate do not modify the kinetic parameters evaluated for nucleoside acceptors. 5. We suggest that the nucleoside phosphotransferase contains both substrate and regulatory sites. It seems that the free apoenzyme is converted, by means of cooperative interactions between regulatory sites, into an enzyme-nucleotide complex, which is particularly stable at 37 degrees C.     

YjeQ,an essential,conserved, uncharacterized protein from Escherichia coli,is an unusual GTPase with circularly permuted G-motifs and marked burst kinetics

The Escherichia coli protein YjeQ represents a protein family whose members are broadly conserved in bacteria and have been shown to be indispensable to the growth of E. coli and Bacillus subtilis [Arigoni, F., et al. (1998) Nat. Biotechnol. 16, 851]. Proteins of the YjeQ family contain all sequence motifs typical of the vast class of P-loop-containing GTPases, but show a circular permutation, with a G4-G1-G3 pattern of motifs as opposed to the regular G1-G3-G4 pattern seen in most GTPases. All YjeQ family proteins display a unique domain architecture, which includes a predicted N-terminal OB-fold RNA-binding domain, the central permuted GTPase module, and a zinc knuckle-like C-terminal cysteine cluster. This domain architecture suggests a possible role for YjeQ as a regulator of translation. YjeQ was overexpressed, purified to homogeneity, and shown to contain 0.6 equiv of GDP. Steady state kinetic analyses indicated slow GTP hydrolysis, with a k(cat) of 9.4 h(-)(1) and a K(m) for GTP of 120 microM (k(cat)/K(m) = 21.7 M(-)(1) s(-)(1)). YjeQ also hydrolyzed other nucleoside triphosphates and deoxynucleotide triphosphates such as ATP, ITP, and CTP with specificity constants (k(cat)/K(m)) ranging from 0.2 to 1.0 M(-)(1) s(-)(1). Pre-steady state kinetic analysis of YjeQ revealed a burst of nucleotide hydrolysis for GTP described by a first-order rate constant of 100 s(-)(1) as compared to a burst rate of 0.2 s(-)(1) for ATP. In addition, a variant in the G1 motif of YjeQ (S221A) was substantially impaired for GTP hydrolysis (0.3 s(-)(1)) with a less significant impact on the steady state rate (1.8 h(-)(1)). In summary, E. coli YjeQ is an unusual, circularly permuted P-loop-containing GTPase, which catalyzes GTP hydrolysis at a rate 45 000 times greater than that of turnover.     

Thermotoga maritima MazG protein has both nucleoside triphosphate pyrophosphohydrolase and pyrophosphatase activities

MazG proteins form a widely conserved family among bacteria, but their cellular function is still unknown. Here we report that Thermotoga maritima MazG protein (Tm-MazG), the product of the TM0913 gene, has both nucleoside triphosphate pyrophosphohydrolase (NTPase) and pyrophosphatase activities. Tm-MazG catalyzes the hydrolysis of all eight canonical ribo- and deoxyribonucleoside triphosphates to their corresponding nucleoside monophosphates and PPi and subsequently hydrolyzes the resultant PPi to Pi. The NTPase activity with deoxyribonucleoside triphosphates as substrate is higher than corresponding ribonucleoside triphosphates. dGTP is the best substrate among the deoxyribonucleoside triphosphates, and GTP is the best among the ribonucleoside triphosphates. Both NTPase and pyrophosphatase activities were enhanced at higher temperatures and blocked by the alpha,beta-methyleneadenosine triphosphate, which cannot be hydrolyzed by Tm-MazG. Furthermore, PPi is an inhibitor for the Tm-MazG NTPase activity. Significant decreases in the NTPase activity and concomitant increases in the pyrophosphatase activity were observed when mutations were introduced at the highly conserved amino acid residues in Tm-MazG N-terminal region (E41Q/E42Q, E45Q, E61Q, R97A/R98A, and K118A). These results demonstrated that Tm-MazG has dual enzymatic functions, NTPase and pyrophosphatase, and that these two enzymatic activities are coordinated.     

Multiple stages in codon-anticodon recognition: double-trigger mechanisms and geometric constraints

Thirty years of kinetic studies on tRNA selection in the elongation cycle are reviewed, and confronted with results derived from various sources, including structural studies on the ribosome, genetic observations on ribosome and EF-Tu accuracy mutants, and codon-specific elongation rates. A coherent framework is proposed, which gives meaning to many puzzling effects. Ribosomal accuracy would be governed by a "double-trigger" principle, according to which the ribosome uses energy in the forward direction to create new configurations for tRNA selection, and energy in the backward direction to regain its initial configuration, in particular after a premature dissociation event. The conformation energy would come in part, in Hopfield's mode, from GTP cleavage on the ternary complex (TC). The reset energy would be provided in part, in the author's mode, from GTP cleavage on a binary EF-Tu.GTP complex (BC). There would be several paths for amino acid incorporation. The path of highest accuracy would involve TC binding followed by BC binding, followed either by GTP hydrolysis on the TC, or by TC dissociation and GTP hydrolysis on the BC. Codon-anticodon recognition would occur in at least three kinetically and geometrically distinct stages. In a first stage, there would be a very rapid sorting of the TCs with unstrained anticodons contacting a loosely held mRNA. This stage ends with the anchoring of the codon-anticodon complex by a cluster of three nucleotides of 16S RNA. The second stage would be the most discriminative one. It would operate on the 5 ms time scale and terminate with GTP cleavage on the TC. The third stage would provide a last, crude selection involving "naked" aa-tRNA, partially held back by steric hindrance. Streptomycin and most EF-Tu mutants as well as high accuracy ribosomal mutants would produce specific alterations at stage 2, which are mapped on the stage 2 kinetic mechanism. The ram ribosomal ambiguity mutants, and anticodon position 37 modifications could be markers of stages 1 and 3 selection. Dissociation events at stage 2 or stage 3, when they are not immediately followed by reset events create a leaky state favorable to shortcut incorporation events. These events are equivalent to an "error-prone codon-anticodon mismatch repair". From the recent evidence on ribosome structure, it is conjectured that the L7/L12 flexible stalk of the large ribosome subunit acts as a proofreading gate, and that the alternation of its GTPase activation center between "TCase" competence and "BCase" competence is a main factor in the control of accuracy.     

Thermodynamic limits on the size and size distribution of nucleic acids synthesized in vitro: the role of pyrophosphate hydrolysis.

The free-energy change of phosphodiester bond formation from nucleoside triphosphates is more favorable than with nucleoside diphosphates as substrates. Base-stacking interactions can make significant contributions to both delta G degrees ' values. Pyrophosphate hydrolysis when it accompanies the former reaction dominates all thermodynamic considerations. Three experimental situations are discussed in which high-molecular-weight polynucleotides are synthesized without a strong driving force for covalent bond formation. For one of these, a kinetic scheme is presented which encompasses an early narrow Poisson distribution of chain lengths with ultimate passage to a disperse equilibrium population of chain sizes. Hydrolytic removal of pyrophosphate expands the time scale for this undesirable process by a factor of 10(9), while it enormously elevates the thermodynamic ceiling for the average degrees of polymerization in the other two examples. The electron micrographically revealed broad size population from an early study of partial replication of a T7 DNA template is found to adhere (fortuitously) to a disperse most probable representation. Some possible origins are examined for the branched structures in this product, as well as in a later investigation of replication of this nucleic acid. The achievement of both very high molecular weights and sharply peaked size distributions in polynucleotides synthesized in vitro will require coupling to inorganic pyrophosphatase action as in vivo.