ATP-binding site of human brain hexokinase as studied by molecular modeling and site-directed mutagenesis

The interaction of ATP with the active site of hexokinase is unknown since the crystal structure of the hexokinase-ATP complex is unavailable. It was found that the ATP binding site of brain hexokinase is homologous to that of actin, heat shock protein hsc70, and glycerol kinase. On the basis of these similarities, the ATP molecule was positioned in the catalytic domain of human brain hexokinase, which was modeled from the X-ray structure of yeast hexokinase. Site-directed mutagenesis was performed to test the function of residues presumably involved in interaction with the tripolyphosphoryl moiety of ATP. Asp532, which is though to be involved in binding the Mg2+ ion of the MgATP2- complex, was mutated to Lys and Glu. The kcat values decreased 1000- and 200-fold, respectively, for the two mutants. Another residue, Thr680 was proposed to interact with the gamma-phosphoryl group of ATP through hydrogen bonds and was mutated to Val and Ser. The kcat value of the Thr680Val mutant decreased 2000-fold, whereas the kcat value of the Thr680Ser decreased only 2.5-fold, implying the importance of the hydroxyl group. The Km and dissociation constant values for either ATP or glucose of all the above mutants showed little or no change relative to the wild-type enzyme. The Ki values for the glucose 6-phosphate analogue 1,5-anhydroglucitol 6-phosphate, were the same as that of the wild-type enzyme, and the inhibition was reversed by inorganic phosphate (Pi) for all four mutants. The circular dichroism spectra of the mutants were the same as that of the wild-type enzyme. The results from the site-directed mutagenesis demonstrate that the presumed interactions of investigated residues with ATP are important for the stabilization of the transition state.     

Location of the phosphate binding site within Clostridium symbiosum pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, Pi, and pyruvate with AMP, PPi, and PEP in three partial reactions: (1) E + ATP --> E.ATP --> E-PP.AMP, (2) E-PP.AMP + Pi --> E-PP.AMP.Pi --> E-P.AMP.PPi, and (3) E-P + pyruvate --> E-P.pyruvate --> E.PEP. The Clostridium symbiosum PPDK structure consists of N-terminal, central, and C-terminal domains. The N-terminal and central domains catalyze partial reactions 1 and 2 whereas the C-terminal and central domains catalyze partial reaction 3. The goal of the present work is to determine where on the N-terminal domain catalysis of partial reactions 1 and 2 occurs and, in particular, where the Pi binding site is located. Computer modeling studies implicated Arg337 as a key residue for Pi binding. This role was tested by site-directed mutagenesis. The R337A PPDK was shown to be impaired in catalysis of the forward (kcat 300-fold lower) and reverse (kcat 30-fold lower) full reactions. Time courses for the single turnover reactions were measured to show that catalysis of partial reaction 1 is 5-fold slower in the mutant, catalysis of the second partial reaction is 140-fold slower in the mutant, and catalysis of the third partial reaction is unaffected. With the exception of the mutation site, the crystal structure of the R337A PPDK closely resembles the structure of the wild-type protein. Thus, the altered kinetic properties observed for this mutant are attributed solely to the elimination of the interaction between substrate and the guanidinium group of the Arg337 side chain. On the basis of these findings we propose that the Pi binding site is located within the crevice of the PPDK N-terminal domain, at a site that is flanked by the ATP beta-P and the Mg2+ cofactor.     

Localization of the active site of type II dehydroquinases. Identification of a common arginine-containing motif in the two classes of dehydroquinases

A novel method based on electrospray mass spectrometry (Krell, T., Pitt, A. R., and Coggins, J. R. (1995) FEBS Lett. 360, 93-96) has been used to localize active site residues in the type I and type II dehydroquinases. Both enzymes have essential hyper-reactive arginine residues, and the type II enzymes have an essential tyrosine residue. The essential hyper-reactive Arg-23 of the Streptomyces coelicolor type II enzyme has been replaced by lysine, glutamine, and alanine residues. The mutant enzymes were purified and shown by CD spectroscopy to be structurally similar to the wild-type enzyme. All three mutant enzymes were much less active, for example the kcat of the R23A mutant was 30,000-fold reduced. The mutants all had reduced Km values, indicating stronger substrate binding, which was confirmed by isothermal titration calorimetry experiments. A role for Arg-23 in the stabilization of a carbanion intermediate is proposed. Comparison of the amino acid sequence around the hyper-reactive arginine residues of the two classes of enzymes indicates that there is a conserved structural motif that might reflect a common substrate binding fold at the active center of these two classes of enzyme.     

Functional evaluation of invariant arginines situated in the mobile lid domain of phosphoribulokinase

Rhodobacter sphaeroides phosphoribulokinase contains four invariant arginines (R49, R168, R173, and R187). The high-resolution structure of this enzyme [Harrison, D. H. T., Runquist, J. A., Holub, A., and Miziorko, H. M. (1998) Biochemistry (submitted for publication)] reveals that it folds in a manner similar to that of adenylate kinase. Three invariant arginines (R168, R173, and R187) as well as arginine-186, which is conserved in prokaryotic phosphoribulokinases, have not been previously functionally evaluated. These arginine residues map within the mobile lid domain that is a distinctive feature of the adenylate kinase family of proteins. Precedent for the significant function of arginines in phosphotransferase reactions prompted substitution of glutamine for each of these three invariant arginines. Solution state characterization of the isolated mutant proteins indicated that they retained a high degree of structural integrity, as indicated by their stoichiometric binding of an alternative nucleotide substrate (trinitrophenyl-ATP) as well as the allosteric effector (NADH). Kinetic characterization indicated > 10(4)-fold diminution in V/KRu5P for R168Q, attributable to a > 300-fold decrease in catalytic efficiency and an increase (approximately 50-fold) in Km Ru5P. For R173Q, a 15-fold diminution in Vmax and a 100-fold increase in Km Ru5P were observed. These observations implicate new components of the ribulose 5-phosphate binding site. Additionally, they confirm assignment of the mobile lid domain as part of the phosphoribulokinase active site, even though this region is well separated from other active site elements in the structure of the open form of the protein. Characterization of R186Q and R187Q mutants suggests that they influence the cooperativity of substrate binding.     

Hydrolysis of phosphodiesters through transformation of the bacterial phosphotriesterase

The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide array of phosphotriesters and related phosphonates, including organophosphate pesticides and military nerve agents. It has now been shown that this enzyme can also catalyze the hydrolysis of phosphodiesters, albeit at a greatly reduced rate. However, the enzymatic hydrolysis of ethyl-4-nitrophenyl phosphate (compound I) by the wild-type enzyme was >10(8) times faster than the uncatalyzed reaction (kcat = 0.06 s-1 and Km = 38 mM). Upon the addition of various alkylamines to the reaction mixture, the kcat/Km for the phosphodiester (compound I) increased up to 200-fold. Four mutant enzymes of the phosphotriesterase were constructed in a preliminary attempt to improve phosphodiester hydrolysis activity of the native enzyme. Met-317, which is thought to reside in close proximity to the pro-S-ethoxy arm of the paraoxon substrate, was mutated to arginine, alanine, histidine, and lysine. These mutant enzymes showed slight improvements in the catalytic hydrolysis of organophosphate diesters. The M317K mutant enzyme displayed the most improvement in catalytic activity (kcat = 0.34 s-1 and Km = 30 mM). The M317A mutant enzyme catalyzed the hydrolysis of the phosphodiester (compound I) in the presence of alkylamines up to 200 times faster than the wild-type enzyme in the absence of added amines. The neutralization of the negative charge on the oxygen atom of the phosphodiester by the ammonium cation within the active site is thought to be responsible for the rate enhancement by these amines in the hydrolytic reaction. These results demonstrate that an active site optimized for the hydrolysis of organophosphate triesters can be made to catalyze the hydrolysis of organophosphate diesters.     

Relationship of conserved residues in the IMP binding site to substrate recognition and catalysis in Escherichia coli adenylosuccinate synthetase

Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme. Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP.     

Function of Pro-185 in the ProCys of conserved motif IV in the EcoRII [cytosine-C5]-DNA methyltransferase

ProCys in the conserved sequence motif IV of [cytosine-C5]-DNA methyltransferases is known to be part of the catalytic site. The Cys residue is directly involved in forming a covalent bond with the C6 of the target cytosine. We have found that substitution of Pro-185 with either Ala or Ser resulted in a reduced rate of methyl group transfer by the EcoRII DNA methyltransferase. In addition, we observed an increase in the Km for substrate S-adenosyl-L-methionine (AdoMet), but a decrease in the Km for substrate DNA. This is reflected in minor changes in kcat/Km for DNA, but in 10- to 100-fold reductions in kcat/Km for AdoMet. This suggests that Pro-185 is important to properly orient the activated cytosine and AdoMet for methyl group transfer by direct interaction with AdoMet and indirectly via the Cys interaction with cytosine.     

Transition state structure of arginine kinase: implications for catalysis of bimolecular reactions

Arginine kinase belongs to the family of enzymes, including creatine kinase, that catalyze the buffering of ATP in cells with fluctuating energy requirements and that has been a paradigm for classical enzymological studies. The 1.86-A resolution structure of its transition-state analog complex, reported here, reveals its active site and offers direct evidence for the importance of precise substrate alignment in the catalysis of bimolecular reactions, in contrast to the unimolecular reactions studied previously. In the transition-state analog complex studied here, a nitrate mimics the planar gamma-phosphoryl during associative in-line transfer between ATP and arginine. The active site is unperturbed, and the reactants are not constrained covalently as in a bisubstrate complex, so it is possible to measure how precisely they are pre-aligned by the enzyme. Alignment is exquisite. Entropic effects may contribute to catalysis, but the lone-pair orbitals are also aligned close enough to their optimal trajectories for orbital steering to be a factor during nucleophilic attack. The structure suggests that polarization, strain toward the transition state, and acid-base catalysis also contribute, but, in contrast to unimolecular enzyme reactions, their role appears to be secondary to substrate alignment in this bimolecular reaction.     

American Board of Emergency Medicine Longitudinal Study of Emergency Physicians

Mutant adenylosuccinate lyases of Bacillus subtilis were prepared by site-directed mutagenesis with replacements for His141, previously identified by affinity labeling as being in the active site [Lee, T. T., Worby, C., Dixon, J. E., and Colman, R. F. (1997) J. Biol. Chem. 272, 458-465]. Four substitutions (A, L, E, Q) yield mutant enzyme with no detectable catalytic activity, while the H141R mutant is about 10(-)5 as active as the wild-type enzyme. Kinetic studies show, for the H141R enzyme, a Km that is only 3 times that of the wild-type enzyme. Minimal activity was also observed for mutant enzymes with replacements for His68 [Lee, T. T., Worby, C., Bao, Z. -Q., Dixon, J. E., and Colman, R. F. (1998) Biochemistry 37, 8481-8489]. Measurement of the reversible binding of radioactive adenylosuccinate by inactive mutant enzymes with substitutions at either position 68 or 141 shows that their affinities for substrate are decreased by only 10-40-fold. These results suggest that His141, like His68, plays an important role in catalysis, but not in substrate binding. Evidence is consistent with the hypothesis that His141 and His68 function, respectively, as the catalytic base and acid. Circular dichroism spectroscopy and gel filtration chromatography conducted on wild-type and all His141 and His68 mutants reveal that none of the mutant enzymes exhibits major structural changes and that all the enzymes are tetramers. Mixing inactive His141 with inactive His68 mutant enzymes leads to striking increases in catalytic activity. This complementation of mutant enzymes indicates that His141 and His68 come from different subunits to form the active site. A tetrameric structure of adenylosuccinate lyase was constructed by homology modeling based on the known structures in the fumarase superfamily, including argininosuccinate lyase, delta-crystallin, fumarase, and aspartase. The model suggests that each active site is constituted by residues from three subunits, and that His141 and His68 come from two different subunits.     

Isotope exchange studies on the Escherichia coli selenophosphate synthetase mechanism

Selenophosphate synthetase, the Escherichia coli selD gene product, is a 37-kDa protein that catalyzes the synthesis of selenophosphate from ATP and selenide. In the absence of selenide, ATP is converted quantitatively to AMP and two orthophosphates in a very slow partial reaction. A monophosphorylated enzyme derivative containing the gamma-phosphoryl group of ATP has been implicated as an intermediate from the results of positional isotope exchange studies. Conservation of the phosphate bond energy in the final selenophosphate product is indicated by its ability to phosphorylate alcohols and amines to form O-phosphoryl- and N-phosphoryl-derivatives. To further probe the mechanism of action of selenophosphate synthetase, isotope exchange studies with [8-14C]ADP or [8-14C]AMP and unlabeled ATP were carried out, and 31P NMR analysis of reaction mixtures enriched in H218O was performed. A slow enzyme-catalyzed exchange of ADP with ATP observed in the absence of selenide implies the existence of a phosphorylated enzyme and further supports an intermediary role of ADP in the reaction. Under these conditions ADP is slowly converted to AMP. Incorporation of 18O from H218O exclusively into orthophosphate in the overall selenide-dependent reaction indicates that the beta-phosphoryl group of the enzyme-bound ADP is attacked by water with liberation of orthophosphate and formation of AMP. Based on these results and the failure of the enzyme to catalyze an exchange of labeled AMP with ATP, the existence of a pyrophosphorylated enzyme intermediate that was postulated earlier can be excluded.     

Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway

A bifunctional enzyme, fructose-6-phosphate,2-kinase/fructose 2, 6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-Pase), catalyzes synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P2). Previously, the rat liver Fru-2,6-Pase reaction (Fru-2,6-P2 --> Fru-6-P + Pi) has been shown to proceed via a phosphoenzyme intermediate with His258 phosphorylated, and mutation of the histidine to alanine resulted in complete loss of activity (Tauler, A., Lin, K., and Pilkis, S. J. (1990) J. Biol. Chem. 265, 15617-15622). In the present study, it is shown that mutation of the corresponding histidine (His256) of the rat testis enzyme decreases activity by less than a factor of 10 with a kcat of 17% compared with the wild type enzyme. Mutation of His390 (in close proximity to His256) to Ala results in a kcat of 12.5% compared with the wild type enzyme. Attempts to detect a phosphohistidine intermediate with the H256A mutant enzyme were unsuccessful, but the phosphoenzyme is detected in the wild type, H390A, R255A, R305S, and E325A mutant enzymes. Data demonstrate that the mutation of His256 induces a change in the phosphatase hydrolytic reaction mechanism. Elimination of the nucleophilic catalyst, H256A, results in a change in mechanism. In the H256A mutant enzyme, His390 likely acts as a general base to activate water for direct hydrolysis of the 2-phosphate of Fru-2,6-P2. Mutation of Arg255 and Arg305 suggests that the arginines probably have a role in neutralizing excess charge on the 2-phosphate and polarizing the phosphoryl for subsequent transfer to either His256 or water. The role of Glu325 is less certain, but it may serve as a general acid, protonating the leaving 2-hydroxyl of Fru-2,6-P2.     

Role of the active-site carboxylate in dihydrofolate reductase: kinetic and spectroscopic studies of the aspartate 26-->asparagine mutant of the Lactobacillus casei enzyme

A mutant of Lactobacillus casei dihydrofolate reductase, D26N, in which the active site aspartic acid residue has been replaced by asparagine by oligonucleotide-directed mutagenesis has been studied by NMR and optical spectroscopy and its kinetic behavior characterized in detail. On the basis of comparisons of a large number of chemical shifts and NOEs, it is clear that there are only very slight structural differences between the methotrexate complexes of the wild-type and mutant enzymes and that these are restricted to the immediate environment of the substitution. The data suggest a slight difference in orientation of the pteridine ring in the binding site in the mutant enzyme. Both NMR and UV spectroscopy show that methotrexate is protonated on N1 when bound to the wild-type enzyme but not when bound to the mutant. Binding constant measurements by fluorescence quenching and steady-state kinetic measurements of dihydrofolate (FH2) and folate reduction show that the substitution has little or no effect on substrate, coenzyme, and inhibitor binding (< 7-fold increase in Kd) and only a modest effect on kcat (up to a factor of 9 for FH2 and 25 for folate) and kcat/KM (up to a factor of 13 for FH2 and 14 for folate). Measurements of deuterium isotope effects and direct measurements of hydride ion transfer and product release by stopped-flow methods revealed that for the mutant enzyme hydride ion transfer is rate-limiting across the pH range 5-8. This allowed a direct comparison of the rate of hydride ion transfer in the wild-type and mutant enzymes; the asparagine substitution was found to decrease this rate by 62-fold at pH 5.5 and 9-fold at pH 7.5. This effect is much smaller than that seen for the corresponding mutant of Escherichia coli dihydrofolate reductase [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science 231, 1123-1128], estimated as a 1000-fold decrease in the rate of hydride ion transfer. The change in pH dependence of kcat resulting from the substitution is consistent with, but does not prove, the idea that the group of pK 6.0 which must be protonated for hydride ion transfer to occur is Asp26. For folate reduction, the pH dependence of kcat is determined by two pKs, one of which, pK 5, disappears in the mutant enzyme, suggesting that it may correspond to ionization of Asp26.(ABSTRACT TRUNCATED AT 400 WORDS)     

The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity

Leukotriene-A4 hydrolase (EC 3.3.2.6) cleaved the NH2-terminal amino acid from several tripeptides, typified by arginyl-glycyl-aspartic acid, arginyl-glycyl-glycine, and arginyl-histidyl-phenylalanine, with catalytic efficiencies (kcat/Km) > or = 1 x 10(6) M-1 s-1. This exceeds by 10-fold the kcat/Km for its lipid substrate leukotriene A4. Catalytic efficiency declined for dipeptides which had kcat/Km ratios 10-100-fold lower than tripeptides. Tetrapeptides and pentapeptides were even poorer substrates with catalytic efficiencies below 10(3) M-1 s-1. The enzyme preferentially hydrolyzed tripeptide substrates and single amino acid p-nitroanilides with L-arginine at the NH2 terminus. Peptides with proline at the second position were not hydrolyzed, suggesting a requirement for an N-hydrogen at the peptide bond cleaved. Peptides with a blocked NH2 terminus were not hydrolyzed. The specificity constant (kcat/Km) was optimal at pH 7.2 with pK values at 6.8 and 7.9; binding was maximal at pH 8.0. Serum albumins activated the peptidase, increasing tripeptide affinities (Km) by 3-10-fold and specificities (kcat/Km) by 4-13-fold. Two known inhibitors of arginine peptidases, arphamenine A and B, inhibited hydrolysis of L-arginine p-nitroanilide with dissociation constants = 2.0 and 2.5 microM, respectively. Although the primary role of LTA4 hydrolase is widely regarded as the conversion of the lipid substrate leukotriene A4 into the inflammatory lipid mediator leukotriene B4, our data are the first showing that tripeptides are "better" substrates. This is compatible with a biological role for the peptidase activity of the enzyme and may be relevant to the distribution of the enzyme in organs like the ileum, liver, lung, and brain. We present a model which accommodates the available data on the interaction of substrates and inhibitors with the enzyme. This model can account for overlap in the active site for hydrolysis of leukotriene A4 and peptide or p-nitroanilide substrates.     

Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-active site residues

Directed evolution was used to change the substrate specificity of aspartate aminotransferase. A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 x 10(6)-fold increase in the catalytic efficiency (kcat/Km) for a non-native substrate, valine. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, is also changed significantly by the mutations. Interestingly, only one of the 17 residues appears to be able to contact the substrate, and none of them interact with the coenzyme. The three-dimensional structure of the mutant enzyme complexed with a valine analog, isovalerate (determined to 2.4-A resolution by x-ray crystallography), provides insights into how the mutations affect substrate binding. The active site is remodeled; the subunit interface is altered, and the enzyme domain that encloses the substrate is shifted by the mutations. The present results demonstrate clearly the importance of the cumulative effects of residues remote from the active site and represent a new line of approach to the redesign of enzyme activity.     

Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase

Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity. To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli. Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria. The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme. The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes. Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue. The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme. Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack. Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions. Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form. Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.     

Use of inducible feedback-resistant N-acetylglutamate synthetase (argA) genes for enhanced arginine biosynthesis by genetically engineered Escherichia coli K-12 strains

The goal of this work was to construct Escherichia coli strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. coli strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33-38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain "arg boxes," which titrate the ArgR repressor protein, with or without the E. coli carAB genes encoding carbamyl phosphate synthetase and the argI gene for ornithine transcarbamylase. The free arginine production rates of "arg-derepressed" E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt) argA. The expression system with fbr argA produced 7- to 35-fold more arginine than a system overexpressing carAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5 alpha strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carAB and argI genes. Plasmids containing wt or fbr argA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.     

Isolation and characterization of the heat-responsive genes in Escherichia coli

Isocitrate lyase from Escherichia coli has been expressed in transformed E. coli JE10 cells lacking the isocitrate lyase (icl) gene. After directed mutagenesis of icl by the restriction-site elimination method, partially purified isocitrate lyase mutants in which His 356 has been converted to Lys, Arg, Gln, Asp, or Leu have been characterized after induction of transformed, induced JE10 cells. Values of kcat compared to those for wild-type (wt) enzyme (100) at 37 degrees C, pH 7.3, are 18, 1, <1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively. Km values for the 1:1 Mg-isocitrate complex (in millimolar units) are: 0.13, wt; 0.11, H356K; and 0.63, H356R. Further chromatographic purification of isocitrate lyase yields highly purified wt, H356K, and H356R enzymes. The pH profile of the stability of isocitrate lyase, which has never been reported, showed that the H356R enzyme was unstable in the pH range investigated; the wt and H356R variant differed but each was sufficiently stable to study the pH dependence of catalysis. The log kcat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pKa and pKb values for dissociation of an ionizable group on the enzyme-substrate complex of <6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes. Plots of pKm vs pH were different for the wt and H356K variant. Values of pKa and pKb (derived from log kcat/Km plots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pKb value for the wt enzyme was 8.4. The data suggest that His 356 is an important functional residue in isocitrate lyase, perhaps in deprotonating isocitrate during catalytic cleavage.     

Random sequence mutagenesis and resistance to 5-fluorouridine in human thymidylate synthases

Thymidylate synthase (TS) catalyzes the methylation of dUMP to dTMP and is the target for the widely used chemotherapeutic agent 5-fluorouracil. We used random sequence mutagenesis to replace 13 codons within the active site of TS and obtain variants that are resistant to 5-fluorodeoxyuridine (5-FdUR). The resulting random library was selected for its ability to complement a TS-deficient Escherichia coli strain, and sequence analysis of survivors found multiple substitutions to be tolerable within the targeted region. An independent selection of the library was carried out in the presence of 5-FdUR, resulting in a more limited spectrum of mutations. One specific mutation, C199L, was observed in more than 46% of 5-FdUR-resistant clones. A 5-FdUR-resistant triple mutant, A197V/L198I/C199F, was purified to apparent homogeneity. Kinetic studies with the substrate dUMP indicate that this mutant is similar to the wild type in regards to kcat and Km values for dUMP and the cosubstrate CH2H4-folate. In contrast, equilibrium binding studies with the inhibitor, FdUMP, demonstrate that the dissociation constant (Kd) for FdUMP binding into the ternary complex was 20-fold higher than values obtained for the wild-type enzyme. This 5-FdUMP-resistant mutant, or others similarly selected, is a candidate for use in gene therapy to render susceptible normal cells resistant to the toxic effects of systemic 5-fluorouracil.     

Toxic mutations in the recA gene of E. coli prevent proper chromosome segregation

The Escherichia coli RecA protein is the prototype of the RecA/RAD51/DMC1 family of strand transferases acting in genetic recombination. The E96D mutant was previously isolated in a screen for toxic recA mutants and was found to constitutively derepress the SOS genes and inhibit chromosome segregation in E. coli. Here, we have found that the E96D mutation lowers the RecA kcat value for ATP hydrolysis 100-fold. Use of this mutant reveals that the ATPase and branch migration activities of RecA are not necessarily required for catalyzing in vivo recombinational pairing and LexA cleavage. In addition to its effect on ATP hydrolysis, the mutation causes ATP to more strongly promote the transition to the biologically active, extended conformation of the RecA enzyme. The enhanced ATP binding is apparently the cause for a broader nucleic acid ligand specificity. The use of RNA and double-stranded DNA as cofactors for LexA cleavage could give rise to the inappropriate, constitutive derepression of the SOS genes. This underscores the need for the ATP affinity to be optimized so that RecA becomes selectively activated only during DNA repair and recombination through binding single-stranded DNA.