Absence Of Cofactors Research Articles

Crystallization and preliminary X-ray diffraction studies of glycerol 3-phosphate cytidylyltransferase from Staphylococcus aureus.

Glycerol 3-phosphate cytidylyltransferase from Staphylococcus aureus (TarD(Sa)) has been expressed in Escherichia coli, purified to homogeneity and crystallized. The strategy used for determining crystallization conditions employed hanging-drop sparse-matrix screens and required a combination of three different optimization approaches. Specifically, the presence or absence of cofactors needed to be surveyed, the effects of small-molecule additives required exploration and the rate of vapour-diffusion had to be varied in order to obtain crystals of TarD(Sa) suitable for diffraction studies. Crystals thus obtained belong to the space group P3(1)21, with unit-cell parameters a = b = 92.2, c = 156.1 A, and contain four TarD(Sa) molecules per asymmetric unit. The resolution limit observed for these crystals using synchrotron radiation is 3.0 A.

Kinetic studies on veratryl alcohol transformation by horseradish peroxidase.

A number of peroxidases, such as lignin peroxidase and manganese peroxidase have proved to be useful for industrial applications. Some studies on the effects of temperature and pH stability have been carried out. It is known that veratryl alcohol increases their stability in the range 28-50 degrees C and is oxidized, leading to veratryl aldehyde formation. Similar results with horseradish peroxidase (HRP) in the presence of cofactors were found, but the oxidation of veratryl alcohol in the absence of cofactors was extremely labile at acid pH and inactivated in a few minutes. Considering the growing industrial application of HRP, knowledge of its stability and denaturation kinetics is required. In this study, horseradish peroxidase pool (HRP-VI) and its isoenzymes HRP-VIII (acid) and HRP-IX (basic) have been shown to catalyze the oxidation of veratryl alcohol to veratryl aldehyde in the presence of hydrogen peroxide at pH 5.8 in the 35-45 degrees C range and in the absence of any cofactors. Heat and pH denaturation experiments in the presence and absence of veratryl alcohol incubation were conducted with HRP-VI and HRP-IX isoenzymes. HRP-IX was the most active isoenzyme acting on veratryl alcohol but HRP-VI was the most stable for the temperature range tested. At 35 degrees C the HRP pool presented decay constant (Kd) values of 5.5 x 10(-2) h(-1) and 1.4 10(-2) h(-1) in the absence and presence of veratryl alcohol, respectively, with an effective ratio of 3.9. These results present a new feature of peroxidases that opens one more interesting application of HRP to industrial processes.

Residue Met156 Contributes to the Labile Enzyme Conformation of Coagulation Factor VIIa

Serine protease activation is typically controlled by proteolytic cleavage of the scissile bond, resulting in spontaneous formation of the activating Ile(16)-Asp(194) salt bridge. The initiating coagulation protease factor VIIa (VIIa) differs by remaining in a zymogen-like conformation that confers the control of catalytic activity to the obligatory cofactor and receptor tissue factor (TF). This study demonstrates that the unusual hydrophobic Met(156) residue contributes to the propensity of the VIIa protease domain to remain in a zymogen-like conformation. Mutation of Met(156) to Gln, which is found in the same position of the highly homologous factor IX, had no influence on the amidolytic and proteolytic activity of TF-bound VIIa. Furthermore, the mutation did not appreciably stabilize the labile Ile(16)-Asp(194) salt bridge in the absence of cofactor. VIIa(Gln156) had increased affinity for TF, consistent with a long range conformational effect that stabilized the cofactor binding site in the VIIa protease domain. Notably, in the absence of cofactor, amidolytic and proteolytic function of VIIa(Gln156) were enhanced 3- and 9-fold, respectively, compared with wild-type VIIa. The mutation thus selectively influenced the catalytic activity of free VIIa, identifying the Met(156) residue position as a determinant for the zymogen-like properties of free VIIa.

Unique Structural and Functional Properties of the ATP-binding Domain of Atypical Protein Kinase C-ι

Atypical protein kinase C-iota (aPKCiota) plays an important role in mitogenic signaling, actin cytoskeleton organization, and cell survival. Apart from the differences in the regulatory domain, the catalytic domain of aPKCiota differs considerably from other known kinases, because it contains a modification within the glycine-rich loop motif (GXGXXG) that is found in the nucleotide-binding fold of virtually all nucleotide-binding proteins including PKCs, Ras, adenylate kinase, and the mitochondrial F1-ATPase. We have used site-directed mutagenesis and kinetic analysis to investigate whether these sequence differences affect the nucleotide binding properties and catalytic activity of aPKCiota. When lysine 274, a residue essential for ATP binding and activity conserved in most protein kinases, was replaced by arginine (K274R mutant), aPKCiota retained its normal kinase activity. This is in sharp contrast to results published for any other PKC or even distantly related kinases like phosphoinositide 3-kinase gamma, where the same mutation completely abrogated the kinase activity. Furthermore, the sensitivity of aPKCiota for inhibition by GF109203X, a substance acting on the ATP-binding site, was not altered in the K274R mutant. In contrast, replacement of Lys-274 by tryptophan (K274W) completely abolished the kinase activity of PKCiota. In accordance with results obtained with other kinase-defective PKC mutants, in cultured cells aPKCiota-K274W acted in a dominant negative fashion on signal transduction pathways involving endogenous aPKCiota, whereas the effect of the catalytically active K274R mutant was identical to the wild type enzyme. In summary, aPKCiota differs from classical and novel PKCs also in the catalytic domain. This information could be of significant value for the development of specific inhibitors of aPKCiota as a key factor in central signaling pathways.

DNA helicase RepA: cooperative ATPase activity and binding of nucleotides.

The steady-state kinetic parameters of the ATPase activity of the homohexameric DNA helicase RepA and the binding of the fluorescent analogue epsilonADP to RepA have been studied. ssDNA stimulates RepA ATPase activity optimally at acidic pH 5.3-6.0. The sigmoidal kinetic curves in both the absence and presence of ssDNA show strong positive cooperativity for ATP hydrolysis, with oligonucleotides longer than 10mer optimal for ssDNA-stimulated ATPase activity. Fluorescence titrations show that, at 25 degrees C and in the absence of DNA, the binding of epsilonADP to RepA is biphasic with three high (K(1) = 1.54 x 10(6) M(-1)) and three low (K(2) = 4.71 x 10(4) M(-)(1)) affinity binding sites differing by 30-40-fold in binding constants. In the absence of cofactors, RepA melts cooperatively at T(m) = 65.8 +/- 0.1 degrees C and is more stable in the presence of ATPgammaS, T(m) = 68.1 +/- 0.2 degrees C (DeltaDeltaG 0.95 kcal/mol), than in the presence of ADP, T(m) = 66. 5 +/- 0.1 degrees C (DeltaDeltaG 0.29 kcal/mol), indicating that the additional phosphate group in ATPgammaS has a significant influence on RepA structure. A model is proposed in which individual subunits of RepA sequentially and cooperatively perform a multistep ATP hydrolytic cycle.

Characterization of trehalose phosphorylase from Schizophyllum commune

During growth on d-glucose, the basidiomycete Schizophyllum commune produces an intracellular alpha,alpha-trehalose phosphorylase. Specific phosphorylase activity increases steadily during the exponential growth phase, up to a maximum of approx. 0.08 unit/mg of protein, and decreases after the available d-glucose in the medium has been fully depleted. The variation with time of the concentrations of intracellular alpha,alpha-trehalose and Pi is reciprocal to that of trehalose phosphorylase activity, indicating that the enzyme makes temporary use of the pool of alpha, alpha-trehalose (approx. 0.42 mmol/g dry cell) via phosphorolysis. The enzyme has been purified, 150-fold, to homogeneity in 55% yield and characterized. It is a monomeric 61 kDa protein, which seems to lack regulation at the level of enzyme activity. The enzyme catalyses the reversible phosphorolysis of alpha,alpha-trehalose into alpha-d-glucose 1-phosphate and alpha-d-glucose in the absence of cofactors, with a catalytic-centre activity at 30 degrees C of 14 s(-1). Double-reciprocal analysis of the initial velocities for trehalose phosphorolysis and synthesis yields intersecting patterns, and no exchange reaction occurs between alpha-d-glucose 1-phosphate and the phosphate analogue arsenate. Therefore trehalose phosphorylase operates by a ternary-complex, rather than a Ping-Pong, kinetic mechanism. The specificity constants (kcat/Km) of phosphate (6000 M(-1).s(-1)) and alpha-d-glucose 1-phosphate (3500 M(-1).s(-1)) compared with those of alpha,alpha-trehalose (161 M(-1).s(-1)) and d-glucose (260 M(-1).s(-1)), together with the inhibition by NaCl, which is competitive with respect to phosphate with a Ki of 67 mM, suggest an important role for ionic enzyme-phosphate interactions in the catalytic mechanism of trehalose phosphorylase. The isolated enzyme requires alpha,alpha-trehalose (0.1-0.3 M) for its conformational stability.

Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation.

Enzyme-based chemical transformations typically proceed with high selectivity under mild conditions, and are becoming increasingly important in the pharmaceutical and chemical industries. Cytochrome P450 monooxygenases (P450s) constitute a large family of enzymes of particular interest in this regard. Their biological functions, such as detoxification of xenobiotics and steroidogenesis, are based on the ability to catalyse the insertion of oxygen into a wide variety of compounds. Such a catalytic transformation might find technological applications in areas ranging from gene therapy and environmental remediation to the selective synthesis of pharmaceuticals and chemicals. But relatively low turnover rates (particularly towards non-natural substrates), low stability and the need for electron-donating cofactors prohibit the practical use of P450s as isolated enzymes. Here we report the directed evolution of the P450 from Pseudomonas putida to create mutants that hydroxylate naphthalene in the absence of cofactors through the 'peroxide shunt' pathway with more than 20-fold higher activity than the native enzyme. We are able to screen efficiently for improved mutants by coexpressing them with horseradish peroxidase, which converts the products of the P450 reaction into fluorescent compounds amenable to digital imaging screening. This system should allow us to select and develop mono- and di-oxygenases into practically useful biocatalysts for the hydroxylation of a wide range of aromatic compounds.

Characterization of the Escherichia coliDamage-independent UvrBC Endonuclease Activity

Incision of damaged DNA templates by UvrBC in Escherichia coli depends on UvrA, which loads UvrB on the site of the damage. A 50-base pair 3' prenicked DNA substrate containing a cholesterol lesion is incised by UvrABC at two positions 5' to the lesion, the first incision at the eighth and the second at the 15th phosphodiester bond. Analysis of a 5' prenicked cholesterol substrate revealed that the second 5' incision is efficiently produced by UvrBC independent of UvrA. This UvrBC incision was also found on the same substrate without a lesion and, with an even higher efficiency, on a DNA substrate containing a 5' single strand overhang. Incision occurred in the presence of ATP or ADP but not in the absence of cofactor. We could show an interaction between UvrB and UvrC in solution and subsequent binding of this complex to the substrate with a 5' single strand overhang. Analysis of mutant UvrB and UvrC proteins revealed that the damage-independent nuclease activity requires the protein-protein interaction domains, which are exclusively needed for the 3' incision on damaged substrates. However, the UvrBC incision uses the catalytic site in UvrC which makes the 5' incision on damaged DNA substrates.

Sequence-specific DNA binding by EcoKI, a type IA DNA restriction enzyme

The type I DNA restriction and modification enzymes of prokaryotes are multimeric enzymes that cleave unmethylated, foreign DNA in a complex process involving recognition of the methylation status of a DNA target sequence, extensive translocation of DNA in both directions towards the enzyme bound at the target sequence, ATP hydrolysis, which is believed to drive the translocation possibly via a helicase mechanism, and eventual endonucleolytic cleavage of the DNA. We have examined the DNA binding affinity and exonuclease III footprint of the EcoKI type IA restriction enzyme on oligonucleotide duplexes that either contain or lack the target sequence. The influence of the cofactors, S-adenosyl methionine and ATP, on binding to DNA of different methylation states has been assessed. EcoKI in the absence of ATP, with or without S-adenosyl methionine, binds tightly even to DNA lacking the target site and the exonuclease footprint is large, approximately 45 base-pairs. The protection is weaker on DNA lacking the target site. Partially assembled EcoKI lacking one or both of the subunits essential for DNA cleavage, is unable to bind tightly to DNA lacking the target site but can bind tightly to the recognition site. The addition of ATP to EcoKI, in the presence of AdoMet, allows tight binding only to the target site and the footprint shrinks to 30 base-pairs, almost identical to that of the modification enzyme which makes up the core of EcoKI. The same effect occurs when S-adenosyl homocysteine or sinefungin are substituted for S-adenosyl methionine, and ADP or ATPγS are substituted for ATP. It is proposed that the DNA binding surface of EcoKI comprises three regions: a “core” region which recognises the target sequence and which is present on the modification enzyme, and a region on each DNA cleavage subunit. The cleavage subunits make tight contacts to any DNA molecule in the absence of cofactors, but this contact is weakened in the presence of cofactors to allow the protein conformational changes required for DNA translocation when a target site is recognised by the core modification enzyme. This weakening of the interaction between the DNA cleavage subunits and the DNA could allow more access of exonuclease III to the DNA and account for the shorter footprint.

Crystallization of Arthrobacter sp. strain 1C N-(1-D-carboxyethyl)-L-norvaline dehydrogenase and its complex with NAD+

The novel NAD+-linked opine dehydrogenase from a soil isolate Arthrobacter sp. strain 1C belongs to an enzyme superfamily whose members exhibit quite diverse substrate specificites. Crystals of this opine dehydrogenase, obtained in the presence or absence of co-factor and substrates, have been shown to diffract to beyond 1.8 Å resolution. X-ray precession photographs have established that the crystals belong to space group P21212, with cell parameters a = 104.9, b = 80.0, c = 45.5 Å and a single subunit in the asymmetric unit. The elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this novel class of dehydrogenases to enable a comparison to be made with other enzyme families and also as the basis for mutagenesis experiments directed towards the production of natural and synthetic opine-type compounds containing two chiral centres.

Application of a Single-Plasmid Vector for Mutagenesis and High-Level Expression of Thioredoxin Reductase and Its Use to Examine Flavin Cofactor Incorporation

Thioredoxin reductase fromEscherichia coliis a dimeric enzyme containing one FAD and one redox-active disulfide per monomer and catalyzes the transfer of electrons from NADPH to thioredoxin, which subsequently performs several important cellular functions. To overcome problems with site-directed mutagenesis and low expression, the thioredoxin reductase gene was adapted for use in the plasmid vector pSL350 (Brosius, J.,Methods Enzymol.216, 469–483, 1992), which is designed both for protein expression and for production of single-stranded template DNA for mutagenesis, and examined expression of wild-type thioredoxin reductase under different growth conditions. In the absence of IPTG inducer, expression of thioredoxin reductase in saturated cultures accounts for 19% of the soluble protein, and with 1 mmIPTG expression increases to 61%. Some of the thioredoxin reductase is expressed as apoenzyme with the amount of apoenzyme increasing at higher IPTG concentrations, accounting for as high as 68% of the total thioredoxin reductase expressed. The apoenzyme in cell extracts is activated rapidly by addition of FAD, indicating correct folding of the enzyme in the absence of cofactor. Purification of wild-type thioredoxin reductase from the new system yielded 189 mg of enzyme from a 300-ml uninduced culture. The new plasmid was also used to generate an N155Y mutant which is purified and partially characterized.

Studies of the esterase activity of cytosolic aldehyde dehydrogenase with resorufin acetate as substrate.

Resorufin acetate is a very good substrate for sheep liver cytosolic aldehyde dehydrogenase, both from the point of view of practical spectrophotometry and in terms of information provided about the nature of the catalysis shown by this enzyme. p-Nitrophenyl (PNP) acetate competes against resorufin acetate for the enzyme's active site (although relatively weakly as the latter substrate has the lower Michaelis constant), but acetaldehyde (in the presence of NAD+) inhibits the hydrolysis of resorufin acetate only at very high aldehyde concentration. In the absence of cofactor, the rate-limiting step in the hydrolysis of resorufin acetate and of PNP acetate is hydrolysis of the common acetyl-enzyme, as shown by the observation of bursts of chromophoric product and very similar values of kcat. In the presence of NAD+ or NADH, however, the deacylation step with resorufin acetate is greatly accelerated until acylation seems to become rate-limiting, because no burst is seen under these conditions. Millimolar concentrations of Mg2+ activate the hydrolyis of resorufin acetate both in the presence and absence of cofactors. With both Mg2+ and cofactor the kcat for hydrolysis of resorufin acetate is 30-35 s-1; this is three orders of magnitude higher than the kcat for aldehyde oxidation in the presence of Mg2+, showing that the enzyme's potential catalytic efficency is very much hampered by the slowness with which NADH dissociates from its binding site. The pH profile for the hydrolysis of resorufin acetate in the presence of NAD+ or NADH fits well to a theoretical ionization curve of pKa approx. 8.2; it is suggested that this might belong to the enzyme's putative catalytic residue (Cys-302).

Roles of Tyr103 and Tyr264 in the regulation of RecA-DNA interactions by nucleotide cofactors.

The DNA-binding mode of the RecA protein, in particular its dependence on nucleotide cofactor, has been investigated by monitoring the fluorescence and linear-dichroism signals of a tryptophan residue inserted in the RecA to replace tyrosine at position 103 or 264. These residues are important for cofactor and DNA binding, as evidenced from their fluorescence changes upon binding of cofactor and DNA [Morimatsu, K., Horii, T. & Takahashi, M. (1995) Eur. J. Biochem. 228, 779-785]. The substitution of these residues with tryptophan does not affect the structure or biological function of the complex and can therefore be exploited to gain structural information in terms of the orientation and environment of the inserted reporter chromophore. The fluorescence change upon formation of the ternary cofactor.RecA. DNA complex was much smaller than the sum of the changes induced by cofactor or DNA alone. This difference indicates that the cofactor and DNA interact with RecA via common components. The fluorescence change caused by DNA in the presence of cofactor was almost independent of the base composition of DNA, in contrast to the interaction in the absence of cofactor. Hence, the contact mode between the selected residues and DNA in the complex may depend significantly on the cofactor. Linear-dichroism measurements indicate that the cofactor does not markedly alter the organization of RecA filament. Linear dichroism shows that neither the aromatic moiety of residue 103 nor that of residue 264 is intercalated between the DNA bases. The textural changes reported for the helical pitch and contour length of RecA fiber upon interaction with cofactor and DNA may derive from a subtle change in orientation of the RecA subunits in the filament.

Cross-linking of SsoII restriction endonuclease to cognate and non-cognate DNAs

Specific and non-specific interactions SsoII restriction endonuclease (R· SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R· SsoII with six N-terminal His residues was shown to be cross-linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R· SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non-cognate DNAs in the absence of cofactor, Mg 2+ ions.

Growth response of adult germ cells to rat androgen-binding protein and human sex hormone-binding globulin.

Androgen-binding protein (ABP) is produced by Sertoli cells depending on the development and the stage of the spermatogenic cycle. Germ cell proliferation is at its peak when ABP is at its peak and secreted towards the testicular basal compartment containing spermatogonia and premeiotic spermatocytes. Rat isolated adult germ cell DNA synthesis was studied in vitro in the presence of ABP with and without steroids and in the presence of pure or recombinant sex steroid hormone-binding globulin (SHBG) using thymidine incorporation. Results are: SHBG is able to promote DNA synthesis in the absence of cofactors. Testosterone reacted negatively to the stimulatory effect of SHBG. We conclude that ABP, the physiological steroid-binding protein, should be considered as a paracrine regulator of spermatogenic DNA synthesis in the adult rat.

Tetrahydrobiopterin deficiency and brain nitric oxide synthase in the hph1 mouse.

Tetrahydrobiopterin (BH4) is the cofactor for the aromatic amino acid monoxygenase group of enzymes and for all known isoforms of nitric oxide synthase (NOS). Inborn errors of BH4 metabolism lead to hyperphenylalaninaemia and impaired catecholamine and serotonin turnover. The effects of BH4 deficiency on brain nitric oxide (NO) metabolism are not known. In this study we have used the hph-1 mouse, which displays GTP cyclohydrolase deficiency, to study the effects of BH4 deficiency on brain NOS. In the presence of exogenous BH4, NOS specific activity was virtually identical in the control and hph-1 preparations. However, omission of BH4 from the reaction buffer led to a significant 20% loss of activity in the hph-1 preparations only. The Km for arginine was virtually identical for the control and hph-1 NOS when BH4 was present in the reaction buffer. In the absence of cofactor, the Km for arginine was 3-fold greater for control and 5-fold greater for hph-1 preparations. It is concluded that (a) BH4 does not regulate the intracellular concentration of brain NOS; (b) less binding of BH4 to NOS occurs in BH4 deficiency states; (c) BH4 has a potent effect on the affinity of NOS for arginine; and (d) the availability of arginine for NOS activity may become severely limiting in BH4 deficiency states. Since, in the presence of suboptimal concentrations of BH4 or arginine, NOS may additionally form oxygen free-radicals, it is postulated that in severe BH4 deficiency states NO formation is impaired and the central nervous system is subjected to increased oxidative stress.

Factor VIIa Residue Arg290 Is Required for Efficient Activation of the Macromolecular Substrate Factor X

The serine protease factor VIIa (VIIa) in complex with tissue factor is responsible for initiating proteolytic events in the coagulation pathways. Efficient proteolysis by the extrinsic activation complex appears to depend on structural determinants in the cofactor as well as the light and heavy chain domains of VIIa. This study characterizes the functional defect resulting from alanine replacement for R290 in the VIIa protease domain. VIIa R290-->A bound both full-length and soluble tissue factor with affinities indistinguishable from wild-type VIIa, consistent with overall unaltered folding of the mutant protein. The catalytic function of VIIa R290-->A was further demonstrated to be unperturbed when analyzed with three different peptidyl p-nitroanilide substrates, indicating that the function of the catalytic triad is not affected by the mutation. However, proteolytic activation of factor X was diminished due to a 4-5-fold decreased kcat in the presence and a > 10-fold decreased rate in the absence of a negatively charged phospholipid surface. The functional defect resulting from the R290-->A replacement was observed in the presence and absence of cofactor. Within the structural framework of serine protease domains, R290 is predicted to be localized in a surface-exposed loop suggested to contribute to substrate selectivity in other serine proteases, consistent with the proposed functional role of R290 in the proteolytic activation of the natural substrate factor X.

The cofactor ATP in DNA-RecA complexes is not intercalated between DNA bases.

In an attempt to understand the role of ATP as a cofactor at the interaction of the RecA protein with DNA, we have studied the orientation geometries of the cofactor analogs adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in RecA-DNA complexes using flow linear dichroism spectroscopy. Both cofactors promote the formation of RecA-DNA complexes of similar structure as judged from similar orientations of DNA bases. The DNA orientation was probed through the dichroism of the long-wavelength absorption of a DNA analog, poly(d epsilon A). In this way differences between the dichroic spectra of the ATP gamma S-RecA-DNA and GTP gamma S-RecA-DNA complexes, observed in the shorter-wavelength region, are related to orientation at variations of the cofactor chromophores. The results show that the guanine plane of GTP gamma S is oriented parallel with the principal axis of the complex in contrast to the more perpendicular orientation of the DNA bases. This observation directly excludes the possibility that the cofactor could be intercalated between the DNA bases. The orientation of the adenine base of ATP gamma S, which may be similar to that of guanine of GTP gamma S albeit not exactly the same, is also inconsistent with intercalation. The possibility that the cofactor bound to the protein could be intercalated in DNA had been speculated from the observation that some DNA intercalators can induce RecA binding to DNA in the absence of cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial restoration of activity to Lactobacillus casei thymidylate synthase following inactivation by domain deletion.

Thymidylate synthase (TS) from Lactobacillus casei has a 50 amino acid insert (residues 90-139) in the small domain that is found in only one other TS. A deletion mutant was constructed which lacked the entire insert, thereby reducing the small domain to the size found in Escherichia coli TS. This mutant did not catalyze the formation of dTMP. From the crystal structure of L. casei TS, we surmised that the loss of activity might have resulted from the exposure of residues of helices C and D, which were previously buried by the insert. To restore the local structure of helices C and D in the deletion mutants, we replaced several residues in this region by the corresponding residues found in E. coli TS. The mutant whose sequence most closely resembled that of E. coli TS carried six mutations and possessed partially restored TS activity. The mutant which had all those mutations except F87D did not catalyze any dTMP formation. The crucial role of F87D was proven in a deletion mutant which had only this change and showed greatly increased activity. All of the mutants catalyzed the debromination of BrdUMP in the absence of cofactor about as well as wild type TS. The kinetic parameters for dTMP formation of the active mutants show that the deletion has its major effect on kcat and binding of cofactor CH2H4folate, with less effect on binding of the substrate dUMP. Removal of residues 90-139 is believed to disorder helices C and D, which in turn decreases cofactor binding and catalysis.

Sulphotransferase-dependent dehydration of atropine and scopolamine in guinea pig.

1. Enzymatic dehydration of atropine and scopolamine was studied in guinea pig. 2. The incubation of these alkaloids with guinea pig liver cytosol in the absence of cofactors gave no dehydrated metabolite. However, when atropine and scopolamine were incubated with cytosol supplemented with ATP and sodium sulphate, dehydrated metabolites, apoatropine and aposcopolamine were formed. The formation of these metabolites was confirmed by gas chromatography-mass spectrometry. 3. The reaction required ATP as well as cytosol as the obligatory factors. Deletion of sodium sulphate from the reaction mixture also resulted in a decrease of the activities, although this treatment showed limited effect when the low concentration of atropine was used. Furthermore, dehydroepiandrosterone, an excellent substrate for hydroxysteroid-sulphotransferase, effectively inhibited the in vitro activity of atropine dehydration. 4. Administration of dehydroepiandrosterone to guinea pig followed by atropine treatment caused decreased urinary excretion of apoatropine. 5. These results strongly suggested that the dehydration of atropine and scopolamine takes place via the sulphate conjugate intermediates produced from the sulphotransferase-catalysed reaction. The present finding is the first example of the sulphotransferase-dependent dehydration of a drug, and its generality in drug metabolism is discussed.