Chemical and kinetic mechanisms of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

The chemical and kinetic mechanisms of purified aspartate-beta-semialdehyde dehydrogenase from Escherichia coli have been determined. The kinetic mechanism of the enzyme, determined from initial velocity, product and dead end inhibition studies, is a random preferred order sequential mechanism. For the reaction examined in the phosphorylating direction L-aspartate-beta-semialdehyde binds preferentially to the E-NADP-Pi complex, and there is random release of the products L-beta-aspartyl phosphate and NADPH. Substrate inhibition is displayed by both Pi and NADP. Inhibition patterns versus the other substrates suggest that Pi inhibits by binding to the phosphate subsite in the NADP binding site, and the substrate inhibition by NADP results from the formation of a dead end E-beta-aspartyl phosphate-NADP complex. The chemical mechanism of the enzyme has been examined by pH profile and chemical modification studies. The proposed mechanism involves the attack of an active site cysteine sulfhydryl on the carbonyl carbon of aspartate-beta-semialdehyde, with general acid assistance by an enzyme lysine amino group. The resulting thiohemiacetal is oxidized by NADP to a thioester, with subsequent attack by the dianion of enzyme bound phosphate. The collapse of the resulting tetrahedral intermediate leads to the acyl-phosphate product and liberation of the active site cysteine.     

Crystallization and preliminary crystallographic analysis of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

Aspartate-beta-semialdehyde dehydrogenase catalyzes the NADPH-mediated reductive dephosphorylation of beta-aspartylphosphate at a branch point in the biosynthesis of several amino acids. The enzyme from Escherichia coli has been crystallized by the vapor diffusion method from Tris buffer (pH 8.5) using polyethylene glycol 4000 as a precipitant. The crystals are orthorhombic and have the symmetry of space group P222(1), with unit cell dimensions of a = 177.8 A, b = 59.9 A, c = 118.65 A, and alpha = beta = gamma = 90 degrees. The dimensions and space group are indicative of two enzyme dimers (40 kDa per subunit) in the asymmetric unit. The crystals show strong diffraction, and a native data set has been collected to 2.5 A resolution.     

L-threonine dehydrogenase from Escherichia coli. Identification of an active site cysteine residue and metal ion studies

Pure L-threonine dehydrogenase from Escherichia coli is a tetrameric protein (Mr = 148,000) with 6 half-cystine residues/subunit; its catalytic activity as isolated is stimulated 5-10-fold by added Mn2+ or Cd2+. The peptide containing the 1 cysteine/subunit which reacts selectively with iodoacetate, causing complete loss of enzymatic activity, has been isolated and sequenced; this cysteine residue occupies position 38. Neutron activation and atomic absorption analyses of threonine dehydrogenase as isolated in homogeneous form now show that it contains 1 mol of Zn2+/mol of enzyme subunit. Removal of the Zn2+ with 1,10-phenanthroline demonstrates a good correlation between the remaining enzymatic activity and the zinc content. Complete removal of the Zn2+ yields an unstable protein, but the native metal ion can be exchanged by either 65Zn2+, Co2+, or Cd2+ with no change in specific catalytic activity. Mn2+ added to and incubated with the native enzyme, the 65Zn2(+)-, the Co2(+)-, or the Cd2(+)- substituted form of the enzyme stimulates dehydrogenase activity to the same extent. These studies along with previously observed structural homologies further establish threonine dehydrogenase of E. coli as a member of the zinc-containing long chain alcohol/polyol dehydrogenases; it is unique among these enzymes in that its activity is stimulated by Mn2+ or Cd2+.     

Mechanism of the reaction catalyzed by isoaspartyl dipeptidase from Escherichia coli

Isoaspartyl dipeptidase (IAD) is a member of the amidohydrolase superfamily and catalyzes the hydrolytic cleavage of beta-aspartyl dipeptides. Structural studies of the wild-type enzyme have demonstrated that the active site consists of a binuclear metal center positioned at the C-terminal end of a (beta/alpha)(8)-barrel domain. Steady-state kinetic parameters for the hydrolysis of beta-aspartyl dipeptides were obtained at pH 8.1. The pH-rate profiles for the hydrolysis of beta-Asp-Leu were obtained for the Zn/Zn-, Co/Co-, Ni/Ni-, and Cd/Cd-substituted forms of IAD. Bell-shaped profiles were observed for k(cat) and k(cat)/K(m) as a function of pH for all four metal-substituted forms. The pK(a) of the group that must be unprotonated for catalytic activity varied according to the specific metal ion bound in the active site, whereas the pK(a) of the group that must be protonated for catalytic activity was relatively independent of the specific metal ion present. The identity of the group that must be unprotonated for catalytic activity was consistent with the hydroxide that bridges the two divalent cations of the binuclear metal center. The identity of the group that must be protonated for activity was consistent with the free alpha-amino group of the dipeptide substrate. Kinetic constants were obtained for the mutant enzymes at conserved residues Glu77, Tyr137, Arg169, Arg233, Asp285, and Ser289. The catalytic properties of the wild-type and mutant enzymes, coupled with the X-ray crystal structure of the D285N mutant complexed with beta-Asp-His, are consistent with a chemical reaction mechanism for the hydrolysis of dipeptides that is initiated by the polarization of the amide bond via complexation to the beta-metal ion of the binuclear metal center. Nucleophilic attack by the bridging hydroxide is facilitated by abstraction of its proton by the side chain carboxylate of Asp285. Collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond occur with donation of a proton from the protonated form of Asp285.     

Mechanism of the physiological reaction catalyzed by tryptophan synthase from Escherichia coli

The physiological synthesis of L-tryptophan from indoleglycerol phosphate and L-serine catalyzed by the alpha 2 beta 2 bienzyme complex of tryptophan synthase requires spatial and dynamic cooperation between the two distant alpha and beta active sites. The carbanion of the adduct of L-tryptophan to pyridoxal phosphate accumulated during the steady state of the catalyzed reaction. Moreover, it was formed transiently and without a lag in single turnovers, and glyceraldehyde 3-phosphate was released only after formation of the carbanion. These and further data prove first that the affinity for indoleglycerol phosphate and its cleavage to indole in the alpha subunit are enhanced substantially by aminoacrylate bound to the beta subunit. This indirect activation explains why the turnover number of the physiological reaction is larger than that of the indoleglycerol phosphate cleavage reaction. Second, reprotonation of nascent tryptophan carbanion is rate limiting for overall tryptophan synthesis. Third, most of the indole generated in the active site of the alpha subunit is transferred directly to the active site of the beta subunit and only insignificant amounts pass through the solvent. Comparison of the single turnover rate constants with the known elementary rate constants of the partial reactions catalyzed by the alpha and beta active sites suggests that the cleavage reaction rather than the transfer of indole or its condensation with aminoacrylate is rate limiting for the formation of nascent tryptophan.     

pH dependency of the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Escherichia coli

The variation with pH of the kinetic parameters associated with the mutase and dehydrogenase reactions catalyzed by chorismate mutase-prephenate dehydrogenase has been determined with the aim of elucidating the role that ionizing amino acid residues play in binding and catalysis. The pH dependency of log V for the dehydrogenase reaction shows that the enzyme possesses a single ionizing group with a pK value of 6.5 that must be unprotonated for catalysis. This same group is observed in the V/Kprephenate, as well as in the V/KNAD, profile. The V/Kprephenate profile exhibits a second ionizing residue with a pK value of 8.4 that must be protonated for the binding of prephenate to the enzyme. For the mutase reaction, the V/Kchorismate profile indicates the presence of three ionizing residues at the active site. Two of these residues, with similar pK values of about 7, must be protonated, while the third, with a pK value of 6.3, must be unprotonated. It can be concluded that all three groups are concerned with the binding of chorismate to the enzyme since the maximum velocity of the mutase reaction is essentially independent of pH. This conclusion is confirmed by the finding that the Ki profile for the competitive inhibitor, (3-endo,8-exo)-8-hydroxy-2-oxabicyclo[3.3]non-6-ene-3,5-dicarboxylic acid, shows the same three ionizing groups as observed in the V/Kchorismate profile. By contrast, the Ki profile for carboxyethyldihydrobenzoate shows only one residue, with a pK value of 7.3, that must be protonated for binding of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoamide dehydrogenase from Escherichia coli. Steady-state kinetics of the physiological reaction

Lipoamide dehydrogenase from Escherichia coli operates qualitatively by the same mechanism as the enzyme from pig heart. It has been suggested that quantitative differences between the two, in particular the marked inhibition of the bacterial enzyme by its product NADH, are related to the fact that the E. coli enzyme lacks the phosphorylation/dephosphorylation control present in the mammalian enzyme (Wilkinson, K. D., and Williams, C. H., Jr. (1981) J. Biol. Chem. 256, 2307-2314). Because of the inhibition by NADH, the kinetics of the E. coli enzyme have not been studied previously in the physiological direction with the natural substrate, dihydrolipoamide. We have now measured the steady-state kinetics of the oxidation of dihydrolipoamide by NAD+ using the stopped-flow technique to follow only the early time course. The pH dependence of kcat revealed an apparent pKa value of 6.7, reflecting ionization(s) of the enzyme-substrate complex. The pH dependence of kcat/Km gave an apparent pKa of 7.4 reflecting ionization(s) of the free 2-electron-reduced enzyme. The inhibition pattern for NADH was mixed, consistent with the fact that NADH is both a product inhibitor and inhibits by reducing a fraction of the enzyme to the catalytically inactive 4-electron-reduced state. There is a modest pH-dependent positive cooperativity in the saturation curve for NAD+ decreasing with increasing pH. Spectral changes in the 530 and 446 nm bands of the 2-electron-reduced enzyme, associated with the titration of the nascent thiols and the base, showed tentative pKa values of 6.4 and 7.1, respectively, in a pH jump experiment. The properties of the wild type E. coli enzyme can now be compared with those of several site-directed mutants.     

The 6-phosphogluconate dehydrogenase reaction in Escherichia coli.

This study is an attempt to relate in vivo use of the 6-phosphogluconate dehydrogenase reaction in Escherichia coli with the characteristics of the enzyme determined in vitro. 1) The enzyme was obtained pure by affinity chromatography and kinetically characterized; as already known, ATP and fructose-1,6-P2 were inhibitors. 2) A series of isogenic strains were made in which in vivo use of thereaction might differ, e.g. a wild type strain versus a mutant lacking 6-phosphogluconate dehydrase, as grown on gluconate; a phosphoglucose isomerase mutant grown on glucose or glycerol. 3) The in vivo rate of use of the 6-phosphogluconate dehydrogenase reaction was determined from measurements of growth rate and yield and from the specific activity of alanine after growth in 1-14C-labeled substrates. 4) The intracellular concentrations of 6-phosphogluconate, NADP+, fructose-1,6-P2, and ATP were measured for the strains in growth on several carbon sources. 5) The metabolite concentrations were used for assay of the enzyme in vitro. The results allow one to calculate how fast the reaction would function in vivo if ATP and fructose-1,6-P2 were its important effectors and if the in vitro assay conditions apply in vivo. The predicted in vivo rates ranged down to as low as one-tenth of the actual rates, and, accordingly, one cannot yet draw firm conclusions about how the reaction is actually controlled in vivo.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 1. Kinetic characterization of the dehydrogenase reaction by use of alternative substrates

The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase-prephenate dehydrogenase, has been isolated from extracts of a plasmid-containing strain of Escherichia coli K12 and purified to homogeneity by a modified procedure that involves chromatography on both Matrex Blue A and Sepharose-AMP. Detailed studies of the dehydrogenase reaction have been undertaken with analogues of prephenate that act as substrates. The analogues, which included two of the four possible diastereoisomers of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate (deoxodihydroprephenate) as well as D- and L-arogenate, were synthesized chemically. As judged by their V/K values, all analogues were poorer substrates than prephenate. The order of their effectiveness as substrates is prephenate greater than one isomer of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate greater than L-arogenate greater than other isomer of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate greater than D-arogenate. Thus the dehydrogenase activity is dependent on the degree and position of unsaturation in the ring structure of prephenate as well as on the type of substitution on the pyruvyl side chain. With prephenate as a substrate, the reaction is irreversible because it involves oxidative decarboxylation. By contrast, 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate undergoes only a simple oxidation, and thus, with this substrate, the reaction is reversible. Steady-state velocity data, obtained by varying substrates over a range of higher concentrations, suggest that the dehydrogenase reaction conforms to a rapid equilibrium, random mechanism with 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate as a substrate in the forward reaction or with the corresponding ketone derivative as a substrate in the reverse direction. The initial velocity patterns obtained by varying prephenate or 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate over a range of lower concentrations, at different fixed concentrations of NAD, were nonlinear and consistent with a unique model that is described by a velocity equation which is the ratio of quadratic polynomials. An equilibrium constant of 1.4 x 10(-7) M for the reaction in the presence of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate indicates that the equilibrium lies very much in favor of ketone production.     

Catalytic cycle of the biosynthetic reaction catalyzed by adenylylated glutamine synthetase from Escherichia coli

The covalently attached AMP moiety of adenylylated glutamine synthetase from Escherichia coli has been replaced by its fluorescent analog, 2-aza-1,N6-etheno-AMP (aza-epsilon-AMP). The modified glutamine synthetase (aza-epsilon-GS) exhibits divalent cation requirement (Mn2+, rather than Mg2+), pH profile, Vmax, and Km similar to those of naturally adenylylated glutamine synthetase. Whereas naturally adenylylated glutamine synthetase exhibits only negligible fluorescence changes upon the binding of substrates, aza-epsilon-GS exhibits large fluorescence changes. The fluorescence changes have been used by means of a stopped flow technique to reveal the involvement of five fluorometrically distinct intermediates in the catalytic cycle for the biosynthesis of glutamine catalyzed by the adenylylated glutamine synthetase. The mechanism is very similar to that previously established for the unadenylylated enzyme, using intrinsic tryptophan fluorescence. Substrates bind via a rapid equilibrium random mechanism, but the reaction proceeds in a stepwise manner. The formation of an enzyme-bound intermediate (probably gamma-glutamyl phosphate + ADP) from ATP and L-glutamate is the rate-limiting step, with the subsequent reaction of the enzyme-bound intermediate occurring very rapidly. The success in elucidating this complex mechanism is due largely to the vastly different amplitudes of the fluorescence changes at the two excitation maxima (300 nm and 360 nm) of the aza-epsilon-AMP moiety which accompany the formation of the various intermediates.     

A novel alpha-proton exchange reaction catalyzed by Escherichia coli methionyl-tRNA synthetase.

The Escherichia coli truncated methionyl-tRNA synthetase (delta MTS) was shown to catalyze alpha-carbon hydrogen-deuterium exchange of L-selenomethionine, L-methionine, L-ethionine, and L-norleucine in the presence of deuterium oxide. The rate of alpha-proton exchange for L-methionine was shown to be linear with respect to delta MTS concentration. The exchange reaction showed saturation kinetics with apparent Km values of 21 and 4 mM in the absence and presence of saturating adenosine concentrations, respectively. As expected, delta MTS did not catalyze alpha-proton exchange of D-methionine since the enzyme has been shown to be specific for L-amino acids. In the absence of enzyme or in the presence of an equivalent concentration of Zn2+, no hydrogen-deuterium exchange was detected. The exchange reaction was not observed with L-methioninol, an analogue of L-methionine lacking the carboxylate group. These results suggest that the alpha-carboxylate group is a requirement for the delta MTS-catalyzed exchange reaction. The E. coli methionyl-tRNA synthetase (MTS) has previously been shown to be a zinc metalloprotein [Posorske, L. H., Cohn, M., Yanagisawa, N., & Auld, D. S. (1979) Biochim. Biophys. Acta 576, 128]. On the basis of the structural and mechanistic information available on MTS, we propose that the enzyme-bound zinc coordinates the carboxylate of the amino acid, while a base on the enzyme is responsible for exchange of the alpha-proton. The role of the enzyme-bound metal is to render the alpha-proton more acidic through coordination of the carboxylate group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.     

Stereochemical course and steady state mechanism of the reaction catalyzed by the GDP-fucose synthetase from Escherichia coli.

Recently the genes encoding the human and Escherichia coli GDP-mannose dehydratase and GDP-fucose synthetase (GFS) protein have been cloned and it has been shown that these two proteins alone are sufficient to convert GDP mannose to GDP fucose in vitro. GDP-fucose synthetase from E. coli is a novel dual function enzyme in that it catalyzes epimerizations and a reduction reaction at the same active site. This aspect separates fucose biosynthesis from that of other deoxy and dideoxy sugars in which the epimerase and reductase activities are present on separate enzymes encoded by separate genes. By NMR spectroscopy we have shown that GFS catalyzes the stereospecific hydride transfer of the ProS hydrogen from NADPH to carbon 4 of the mannose sugar. This is consistent with the stereospecificity observed for other members of the short chain dehydrogenase reductase family of enzymes of which GFS is a member. Additionally the enzyme is able to catalyze the epimerization reaction in the absence of NADP or NADPH. The kinetic mechanism of GFS as determined by product inhibition and fluorescence binding studies is consistent with a random mechanism. The dissociation constants determined from fluorescence studies indicate that the enzyme displays a 40-fold stronger affinity for the substrate NADPH as compared with the product NADP and utilizes NADPH preferentially as compared with NADH. This study on GFS, a unique member of the short chain dehydrogenase reductase family, coupled with that of its recently published crystal structure should aid in the development of antimicrobial or anti-inflammatory compounds that act by blocking selectin-mediated cell adhesion.     

Kinetic studies on the reaction catalyzed by polynucleotide kinase from phage T4-infected Escherichia coli.

Kinetic properties of polynucleotide kinase (EC 2.7.1.78) isolated from Escherichia coli cells infected with phage T4 were investigated. The reaction depends on the concentration of MgATP, while free ATP or free Mg2+ have neither inhibitory nor accelerating effect. The initial reaction velocity was plotted against variable concentrations of ATP as the phosphate donor at various fixed concentrations of 5'-hydroxyl-DNA or -oligo(rA) as the phosphate acceptor in the presence or absence of products. The double reciprocal plot analysis of the data suggested that the reaction obeys the random sequential mechanism. Various constants were determined and the reaction mechanism was discussed.     

Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I.

DNA helicase I, encoded on the Escherichia coli F plasmid, catalyzes a site- and strand-specific nicking reaction within the F plasmid origin of transfer (oriT) to initiate conjugative DNA strand transfer. The product of the nicking reaction contains a single phosphodiester bond interruption as determined by single-nucleotide resolution mapping of both sides of the nick site. This analysis has demonstrated that the nick is located at precisely the same site previously shown to be nicked in vivo (T. L. Thompson, M. B. Centola, and R. C. Deonier, J. Mol. Biol. 207:505-512, 1989). In addition, studies with two oriT point mutants have confirmed the specificity of the in vitro reaction. Characterization of the nicked DNA product has revealed a modified 5' end and a 3' OH available for extension by E. coli DNA polymerase I. Precipitation of nicked DNA with cold KCl in the presence of sodium dodecyl sulfate suggests the existence of protein covalently attached to the nicked DNA molecule. The covalent nature of this interaction has been directly demonstrated by transfer of radiolabeled phosphate from DNA to protein. On the basis of these results, we propose that helicase I becomes covalently bound to the 5' end of the nicked DNA strand as part of the reaction mechanism for phosphodiester bond cleavage. A model is presented to suggest how helicase I could nick the F plasmid at oriT and subsequently unwind the duplex DNA to provide single-stranded DNA for strand transfer during bacterial conjugation.     

Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.     

Solvent isotope effect on the reaction catalysed by the pyruvate dehydrogenase complex from Escherichia coli

The pyruvate dehydrogenase from Escherichia coli showed a primary kinetic isotope effect when its overall reaction or the partial reaction of the pyruvate dehydrogenase component were tested in deuterium oxide. The Michaelis constants for pyruvate were nearly unchanged, but the maximum velocities in water and deuterium oxide differed, their ratio being DV = 1.7 for the overall reaction and DV = 2.1 for the E1p reaction. The pH profile and, accordingly, the delta pK1 and delta pK2 values were shifted by 0.6 units to higher pL values. A linear proton inventory curve was obtained when varying the atom fractions of protons relative to deuterons from 100 to 0%. This is an indication for a single proton transfer. It is proposed that this relatively weak primary isotope effect may be caused by the protonation of the N1' nitrogen at the pyrimidine ring of the cofactor by an adjacent glutamate residue. The proton of its carboxylic group exchanges very fast with deuterons of the solvent.     

Protein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease.

An examination has been made into the nature of the nucleoprotein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease when acting on a pyrimidine dimer-containing fd RF-I DNA species. The complexes of proteins and DNA form in unique stages. The first stage of binding involves an ATP-stimulated interaction of the UvrA protein with duplex DNA containing pyrimidine dimer sites. The UvrB protein significantly stabilizes the UvrA-pyrimidine dimer containing DNA complex which, in turn, provides a foundation for the binding of UvrC to activate the UvrABC endonuclease. The binding of one molecule of UvrC to each UvrAB-damaged DNA complex is needed to catalyze incision in the vicinity of pyrimidine dimer sites. The UvrABC-DNA complex persists after the incision event suggesting that the lack of UvrABC turnover may be linked to other activities in the excision-repair pathway beyond the initial incision reaction.