A once-monthly GLP-1 receptor agonist for treatment of diabetic cats.

There is growing evidence that peptidic glucagon-like peptide-1 receptor agonists (GLP-1RA), such as exenatide, may provide useful therapeutic options for treatment of feline diabetes. However, because such drugs are administered subcutaneously, it is desirable that they be long-acting and not require frequent injections. We have developed a chemically controlled delivery system to support half-life extension of peptidic therapeutics. Here, the peptide is covalently attached to hydrogel microspheres by a self-cleaving ß-eliminative linker; after subcutaneous injection of the microspheres, the peptide is slowly released from the depot to the systemic circulation. Using this technology, we developed a delivery system that supports once-monthly administration of a stable exenatide analog, [Gln28]exenatide, in rodents (Schneider, et al, ACS Chem Biol 12, 2107 to 2116, 2017). The purposes of the present study were a) to demonstrate pharmacokinetic and pharmacodynamic similarities of the deamidation-sensitive GLP-1RA exenatide and the closely related, more stable [Gln28]exenatide and b) to develop a long-acting GLP-1RA in cats. The results show that exenatide and [Gln28]exenatide injected intravenously or subcutaneously at 10 µg/kg have nearly identical pharmacokinetics in the cat-both having elimination half-lives of ∼40 min-but subcutaneously administered [Gln28]exenatide has superior bioavailability-93% for [Gln28]exenatide vs 52% for exenatide. The results also show that exenatide and [Gln28]exenatide have similar insulinotropic activities in the cat during a high-dose intravenous glucose tolerance test; they increased the area under the curve (AUC) for insulin to a similar extent but had no effect on glucose AUC. Finally, subcutaneous injection of a microsphere-[Gln28]exenatide conjugate containing an appropriate self-cleaving linker in the cat provides plasma [Gln28]exenatide with a half-life of about 40 d vs 40 min with the injected free peptide. Hence, the large body of information available for exenatide can be used to facilitate clinical development of [Gln28]exenatide as a treatment for feline diabetes, and the microsphere-[Gln28]exenatide conjugate is quite suitable for once-monthly subcutaneous administration of the peptide in the cat.

Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations.

Cassette replacement of acyltransferase (AT) domains in 6-deoxyerythronolide B synthase (DEBS) with heterologous AT domains with different substrate specificities usually yields the predicted polyketide analogues. As reported here, however, several AT replacements in module 4 of DEBS failed to produce detectable polyketide under standard conditions, suggesting that module 4 is sensitive to perturbation of the protein structure when the AT is replaced. Alignments between different modular polyketide synthase AT domains and the Escherichia coli fatty acid synthase transacylase crystal structure were used to select motifs within the AT domain of module 4 to re-engineer its substrate selectivity and minimize potential alterations to protein folding. Three distinct primary regions of AT4 believed to confer specificity for methylmalonyl-CoA were mutated into the sequence seen in malonyl-CoA-specific domains. Each individual mutation as well as the three in combination resulted in functional DEBSs that produced mixtures of the natural polyketide, 6-deoxyerythronolide B, and the desired novel analogue, 6-desmethyl-6-deoxyerythronolide B. Production of the latter compound indicates that the identified sequence motifs do contribute to AT specificity and that DEBS can process a polyketide chain incorporating a malonate unit at module 4. This is the first example in which the extender unit specificity of a PKS module has been altered by site-specific mutation and provides a useful alternate method for engineering AT specificity in the combinatorial biosynthesis of polyketides.

Synthesis of 14,15-dehydroerythromycin A ketolides: effects of the 13-substituent on erythromycin tautomerism.

A ketolide was prepared from 14,15-dehydroerythromycin A by two different routes. The first approach involving oxidation of the 3-OH of 3-descladinosyl-14,15-dehydroerythromycin A 2'-O-acetate gave unexpectedly high levels of 3,11-double oxidation. This may be due to greater formation of the 9,12-hemiketal in 14,15-dehydroerythromycin A and concomitant exposure of the 11-OH group for oxidation. NMR studies of 14,15-dehydroerythromycin A support this hypothesis, revealing a 9:1 ratio of 9-ketone to 9,12-hemiketal in CDCl3 and a 1:1 ratio in CD3OD as contrasted with the corresponding tautomer ratios of 30:1 in CDCl3, and 6: 1 in CD3OD with erythromycin A. Alteration of the 13-substituent on the erythronolide A ring from ethyl to vinyl thus favors formation of the 9,12-hemiketal. A second route to the ketolides was developed based on these findings, in which the 11-OH is eliminated prior to oxidation of the 3-OH.

Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein.

The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been observed. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as soluble proteins. The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide. Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteins contain a mixture of [3Fe-4S] and [4Fe-4S] cluster states. Reduction with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S](1+) cluster with the majority of the protein containing a species consistent with an S = 0 [4Fe-4S](2+) cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chemistry.

Manipulation of polyketide biosynthesis for new drug discovery.

Modular polyketide synthases (PKS) are large multifunctional proteins which direct the condensation of activated short chain carboxylic acids into products of defined length and functionality using a dedicated set of active sites, or module, for each step in the polymerization. The structure of the product is directly related to the number, content and sequence of modules in a PKS. Technology is described which allows the rational manipulation of the biosynthesis of these compounds and enables the generation of specific novel polyketide structures. Examples of polyketide drugs whose structures may be manipulated using this technology are given.

Physical and biological properties of cationic triesters of phosphatidylcholine.

The properties of a new class of phospholipids, alkyl phosphocholine triesters, are described. These compounds were prepared from phosphatidylcholines through substitution of the phosphate oxygen by reaction with alkyl trifluoromethylsulfonates. Their unusual behavior is ascribed to their net positive charge and absence of intermolecular hydrogen bonding. The O-ethyl, unsaturated derivatives hydrated to generate large, unilamellar liposomes. The phase transition temperature of the saturated derivatives is very similar to that of the precursor phosphatidylcholine and quite insensitive to ionic strength. The dissociation of single molecules from bilayers is unusually facile, as revealed by the surface activity of aqueous liposome dispersions. Vesicles of cationic phospholipids fused with vesicles of anionic lipids. Liquid crystalline cationic phospholipids such as 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine triflate formed normal lipid bilayers in aqueous phases that interacted with short, linear DNA and supercoiled plasmid DNA to form a sandwich-structured complex in which bilayers were separated by strands of DNA. DNA in a 1:1 (mol) complex with cationic lipid was shielded from the aqueous phase, but was released by neutralizing the cationic charge with anionic lipid. DNA-lipid complexes transfected DNA into cells very effectively. Transfection efficiency depended upon the form of the lipid dispersion used to generate DNA-lipid complexes; in the case of the O-ethyl derivative described here, large vesicle preparations in the liquid crystalline phase were most effective.

Characterization of the macrolide P-450 hydroxylase from Streptomyces venezuelae which converts narbomycin to picromycin.

The post-polyketide synthase (PKS) biosynthetic tailoring of macrolide antibiotics usually involves one or more oxidation reactions catalyzed by cytochrome P450 monooxygenases. As the specificities of members from this class of enzymes vary significantly among PKS gene clusters, the identification and study of new macrolide P450s are important to the growing field of combinatorial biosynthesis. We have isolated the cytochrome P450 gene picK from Streptomyces venezuelae which is responsible for the C-12 hydroxylation of narbomycin to picromycin. The gene was located by searching regions proximal to modular PKS genes with a probe for macrolide P450 monooxygenases. The overproduction of PicK with a C-terminal six-His affinity tag (PicK/6-His) in Escherichia coli aided the purification of the enzyme for kinetic analysis. PicK/6-His was shown to catalyze the in vitro C-12 hydroxylation of narbomycin with a kcat of 1.4 s-1, which is similar to the value reported for the related C-12 hydroxylation of erythromycin D by the EryK hydroxylase. The unique specificity of this enzyme should be useful for the modification of novel macrolide substrates similar to narbomycin, in particular, ketolides, a promising class of semisynthetic macrolides with activity against erythromycin-resistant pathogens.

Stereochemical assignment of chiral phosphotriester analogues with Alu I sites.

The stereochemistry of the diastereomers of a DNA duplex with the 2,2,2-trichloro-1,1,-dimethylethyl (TCDME) phosphotriester backbone substitution has been assigned by the use of 2D NMR spectroscopy. The duplex [G1G2A3A4G5p(TCDME)C6T7A8G9G10]-[C11C12T13A14G15C16 T17T18C19C20] is a substrate of the restriction endonuclease Alu I, with placement of the TCDME group at the G5-C6 cleavage site of one strand. The stereochemical orientation of the TCDME group in relation to the structure of the double helix regulates the ability of Alu I to hydrolyze the complementary recognition site. The phosphotriester group of the isomer 1 duplex blocks cleavage of the complementary strand, while that of the isomer 2 duplex allows cleavage to proceed. Within the phosphotriester recognition site, no hydrolysis is detected nor is any seen when the single-stranded DNA substrate is utilized. Data from the 2D NOESY spectra demonstrate that both DNA duplexes retain basic B-form geometry. The isomer 1 duplex shows NOE cross-relaxation from the protons of the two methyl groups of the TCDME modification (1.99, 2.00 ppm) to the G5 H3'(5.30 ppm), G5 H4' (4.53 ppm), and C6 H5'/H5" (4.52, 4.62 ppm) protons. The isomer 2 duplex shows NOE cross-relaxation from the methyl protons of the TCDME modification (2.01, 2.03 ppm) to the C6 H6 (7.15 ppm), C6 H4' (4.30 ppm), C6 H5'/H5" (4.48, 4.62 ppm), G5 H3' (5.26 ppm), and G5 H4' (4.48 ppm) protons. Thus the NOE cross-relaxation between the methyl protons of the TCDME modification and the C6 H6 and C6 H4' protons in isomer 2 is not found in the spectra of the isomer 1 duplex. These NMR data confirm the stereochemical assignment of the chirality of the TCDME phosphotriester group in isomer 1 as the Sp configuration and in isomer 2 as the Rp configuration. The Sp isomer features the TCDME group pointing away from the helix, while the Rp isomer shows the TCDME group pointing towards the major groove. Thus through the use of 2D NMR techniques, the stereochemistry of chiral phosphotriester linkages may be assigned in chemically modified DNA.

Conformational properties of DNA hairpins with TTT and TTTT loops.

The DNA hairpins d[CGATCG-Tn-CGATCG] (n = 3, 4) have been studied by NMR in order to gain information on hairpin conformation and flexibility. Resonance assignments were made using a combination of DQF-COSY, DQF-COSY[31P], NOESY, and 1H-31P-COSY. These data also provide approximate coupling constant information which points out exceptionally flexible regions of the phosphate backbone. The data for both hairpins reveal substantial flexibility within the loop segments. For n = 4, NOESY data alone are insufficient to distinguish between two loop-folding motifs, although coupling constant data favor a conformation in which Tb is folded toward the minor groove and is highly exposed to solvent. This is in agreement with chemical shift data and susceptibility to modification by KMnO4. The phosphate backbone between Tc and Td is exceptionally flexible, undergoing a facile exchange between (beta t,gamma+) and (beta+,gamma t) conformers. A similar flexible phosphate is observed between Tc and C7 when n = 3. Differences in stem conformation and dynamics in both hairpins are restricted to the two base pairs adjacent to the stem-loop junction. The C7pG8 stem phosphate appears to flip easily between (zeta-,alpha-) and (zeta-,alpha t) conformers when n = 4 but not when n = 3. Hairpin loop size thus affects the conformational flexibility of the adjacent stem segment.

Substrate-induced activation of dienelactone hydrolase: an enzyme with a naturally occurring Cys-His-Asp triad.

The Cys-His-Asp catalytic triad found in dienelactone hydrolase (DLH) is unusual for several reasons. It has not been observed in other hydrolytic enzymes and it is virtually inactive when it is produced by site-directed mutagenesis in the proteases. We propose a model to explain why this triad is catalytically active in DLH but not in the proteases. In the resting state of DLH, His202 forms an ion pair with Asp171 and Cys123 exists as a thiol. The resting state thiol does not interact with His202 in the active site but instead forms a hydrogen bond with Glu36 in the interior of the molecule. In the absence of substrate, Glu36 is also ion paired with Arg206. When substrate binds, Arg206 forms a second ion pair with the anionic substrate and the Arg206/Glu36 ion pair weakens. The destabilized Glu36 carboxylate shifts towards and deprotonates the Cys123 thiol, thereby activating the nucleophile. As the thiolate anion is not energetically favoured in the hydrophobic interior of the enzyme, it swings into the active site where it can be stabilized by the His202 imidazolium and the dipole of helix C. The Cys123 thiolate which now lies adjacent to the acyl carbon of the substrate, is thus generated only in the presence of substrate. The mode of thiolate activation reduces the susceptibility of DLH towards thiol alkylating agents.

Catalysis by dienelactone hydrolase: a variation on the protease mechanism.

Dienelactone hydrolase (DLH), an enzyme from the beta-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation.

Biosynthesis of lipoic acid: characterization of the lipoic acid auxotrophs Escherichia coli W1485-lip2 and JRG33-lip9.

The abilities of the Escherichia coli lipoic acid auxotrophs W1485-lip2 and JRG33-lip9 to grow on succinate medium in the presence of octanoate, 8-mercaptooctanoate, or 6-mercaptooctanoate have been determined. Both organisms are mutated in lipA. Neither organism can use octanoate or 6-mercaptooctanoate for production of lipoate, but the lip2 allele can use 8-mercaptooctanoate. Chromosomal DNA from the auxotrophs was amplified by PCR using primers derived from the DNA sequence of wild-type lipA and then sequenced. Both mutants contain single G/C to A/T mutations in lipA, resulting in conversion of Ser307 into Phe in W1485-lip2 and Glu195 into Lys in JRG33-lip9. These results support the hypothesis that lipA is involved in the sulfur insertion step(s) of lipoate biosynthesis and indicate that it is possible to selectively block formation of the C8-S bond through suitable mutation in lipA.

Selective abstraction of 2H from C-1' of the C residue in AGC.ICT by the radical center at C-2 of activated neocarzinostatin chromophore: structure of the drug/DNA complex responsible for bistranded lesion formation.

Glutathione-activated neocarzinostatin chromophore (NCS-Chrom) generates bistranded lesions at AGC.GCT sequences in DNA, consisting of an abasic site at the C residue and a strand break at the T residue on the complementary strand, due to hydrogen atom abstraction from C-1' and C-5', respectively. Earlier work showed that 2H from C-5' of T was selectively abstracted by the radical center at C-6 of activated NCS-Chrom, supporting a proposed model of the active-drug/DNA complex. However, since under the conditions used breaks at the T exceeded their inclusion in bistranded lesions, it was not clear what fraction of the hydrogen transfer represented bistranded lesions. Since virtually all abasic sites at the C are part of a bistranded lesions, hydrogen transfer from C-1' of C into the drug should reflect only the bistranded reaction. Accordingly, a self-complementary oligodeoxynucleotide 5'-GCAGCICTGC-3' was synthesized in which the C contained 2H at the C-1' position. In order to eliminate an 2H isotope effect on the transfer and to increase the extent of the bistranded reaction, an I residue was substituted for the G opposite the C residue. Sequencing gel electrophoretic analysis revealed that under one-hit kinetics, 37% of the damage reaction was associated with abasic site (alkali-labile break) formation at the C residue and 48% with direct strand breaks at the T residue. Thus, 74% of the damage involved a bistranded lesion. 1H NMR spectroscopic analysis of the reacted chromophore showed that 2H had been selectively transferred into the C-2 position to the extent of approximately 22%.(ABSTRACT TRUNCATED AT 250 WORDS)

The biosynthesis of lipoic acid. Cloning of lip, a lipoate biosynthetic locus of Escherichia coli.

The lip gene of Escherichia coli has been cloned and sequenced. Subcloning of a 3-kilobase EcoRI/EcoRV restriction fragment from Clark-Carbon plasmid pLC15-5 into pUC18 gives a plasmid that complements two lipoate auxotrophs, W1485-lip2 and JRG33-lip9, and which expresses a protein of approximately 36,000 Da. Sequencing suggests that lip codes for a protein of 281 amino acids (31,350 Da), showing sequence similarity to biotin synthase. It is thus likely that lip encodes a sulfur insertion enzyme analogous to biotin synthase and that the sulfur insertion chemistries of the two systems are related. Unidirectional nested deletion experiments show that both lipoate auxotrophs are complemented by the same 500-base pair region at the 3' terminus of the lip gene, indicating that the mutations affecting lipoate biosynthesis are located in this region of the protein. A small open reading frame located immediately downstream of the lip gene codes for a small protein of unknown function.

Modulation of neocarzinostatin-mediated DNA double strand damage by activating thiol: deuterium isotope effects.

The neocarzinostatin chromophore causes double-strand damage at AGC sequences on DNA by concomitant 1'-oxidation at C and 5'-oxidation at the T on the complementary strand. The extent of this damage is dependent upon the structure of the thiol used for activation. Deuterium isotope effects suggest that this dependence on thiol structure may be due to internal quenching of one radical site of the activated chromophore by the hydrogen atoms of the thiol sidechain. The 12-mer d[GCAAGCGCTTGC] is treated with the neocarzinostatin chromophore and either sodium thioglycolate or [2-2H2]-thioglycolate, and the distribution of strand breaks is determined by gel electrophoresis. Two isotope effects are noted: an overall sequence-independent effect in which deuterated thioglycolate increases total strand damage by a factor of 2, and a sequence-specific effect by which deuteration increases the proportion of alkali-sensitive strand damage at C6 by an additional factor of 1.5. Methyl thioglycolate shows essentially identical behavior to that of thioglycolate anion, ruling out electrostatic effects as major contributors to the effect of thiol structure on the mode of DNA damage observed. A model for NCSC action consistent with these results is discussed.

Chemical synthesis of oligodeoxynucleotide dumbbells.

The chemical synthesis of DNA dumbbells is investigated by using two sequences, cyclo-d(GCG-T4-CGCCGC-T4-GCG) and cyclo-d(TTCC-T4-GGAATTCC-T4-GGAA). This method readily and inexpensively yields multimicromole quantities of circular DNA, allowing detailed structural and physical studies to be carried out. Linear oligomers of sequence d(GCG-T4-CGCCGC-T4-GCG) having either 3'- or 5'-phosphates were cyclized in 40% and 67% isolated yield, respectively, by using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide. Formation of the circular product is confirmed by a 28 degrees C increase in the optical melting temperature, anomalously rapid electrophoretic migration, sequential nuclear Overhauser enhancements between protons of G1 and G20, and observed nuclear coupling between the ligated phosphorus and protons of both G1 and G20. cyclo-d(TTCC-T4-GGAATTCC-T4-GGCC) was synthesized from the corresponding linear 3'-phosphate in 80% yield by the same procedure. Chemical ligation is most effective for 3'-phosphorylated nick sites flanked by two purine bases.

Inactivation of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii by 2'-chloro-2'-deoxyuridine 5'-triphosphate: a 3'-2' hydrogen transfer during the formation of 3'-keto-2'-deoxyuridine 5'-triphosphate.

The ribonucleoside triphosphate reductase of Lactobacillus leichmannii converts the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP) into a mixture of 2'-deoxyuridine triphosphate (dUTP) and the unstable product 3'-keto-2'-deoxyuridine triphosphate (3'-keto-dUTP). This ketone can be trapped by reduction with NaBH4, producing a 4:1 mixture of xylo-dUTP and dUTP. When [3'-3H]ClUTP is treated with enzyme in the presence of NaBH4, the isomeric deoxyuridines isolated after alkaline phosphatase treatment retained 15% of the 3H in ClUTP. Degradation of these isomeric nucleosides has established the location of the 3H in 3'-keto-dUTP as predominantly 2'(S). The xylo-dU had 98.6% of its label at the 2'(S) position and 1.5% at 2'(R). The isolated dU had 89.6% of its label at 2'(S) and 1.4% at 2'(R), with the remaining 9% label inferred to be at the 3'-carbon, this resulting from the direct enzymic production of dUTP. These results are consistent with enzymic production of a 1:1000 mixture of dUTP and 3'-keto-dUTP, where the 3'-hydrogen of ClUTP is retained at 3' during production of dUTP and is transferred to 2'(S) during production of 3'-keto-dUTP. The implications of these results and the unique role of the cofactor adenosylcobalamin (Ashley et al., 1986) are discussed in terms of reductase being a model for the B12-dependent rearrangement reactions.

Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation.

The ribonucleoside triphosphate reductase (RTPR) of Lactobacillus leichmannii is inactivated by the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP). Inactivation is due to alkylation by 2-methylene-3(2H)-furanone, a decomposition product of the enzymic product 3'-keto-2'-deoxyuridine triphosphate. The former has been unambiguously identified as 2-[(ethylthio)methyl]-3(2H)-furanone, an ethanethiol trapped adduct, which is identical by 1H NMR spectroscopy with material synthesized chemically. Subsequent to rapid inactivation, a slow process occurs that results in formation of a new protein-associated chromophore absorbing maximally near 320 nm. The terminal stages of the inactivation have now been investigated in detail. The alkylation and inactivation stoichiometries were studied as a function of the ratio of ClUTP to enzyme. At high enzyme concentrations (0.1 mM), 1 equiv of [5'-3H]ClUTP resulted in 0.9 equiv of 3H bound to protein and 83% inactivation. The amount of labeling of RTPR increased with increasing ClUTP concentration up to the maximum of approximately 4 labels/RTPR, yet the degree of inactivation did not increase proportionally. This suggests that (1) RTPR may be inactivated by alkylation of a single site and (2) decomposition of 3'-keto-dUTP is not necessarily enzyme catalyzed. The formation of the new protein chromophore was also monitored during inactivation and found to reach its full extent upon the first alkylation. Thus, out of four alkylation sites, only one appears capable of undergoing the subsequent reaction to form the new chromophore. While chromophore formation was prevented by NaBH4 treatment, the chromophore itself is resistant to reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Location of the redox-active thiols of ribonucleotide reductase: sequence similarity between the Escherichia coli and Lactobacillus leichmannii enzymes.

The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with [1-14C]iodoacetamide. The dithiothreitol-reduced E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of 14C. Sequencing of tryptic peptides shows that 2.8 equiv of 14C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of 14C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of 14C. Sequencing of tryptic peptides shows that 1.4 equiv of 14C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I.

2'-Deoxy-2'-halonucleotides as alternate substrates and mechanism-based inactivators of Lactobacillus leichmannii ribonucleotide reductase.

The interaction of the ribonucleoside-triphosphate reductase of Lactobacillus leichmannii with various 2'-halogenated ribo- and arabinonucleoside triphosphates has been investigated. All analogues examined acted as mechanism-based inactivators of the enzyme, producing base, triphosphate, and halide. In all cases, the inactive enzyme had developed the distinctive chromophore at 320 nm that is characteristic of enzyme inactivated by 2-methylene-3(2H)-furanone. The striking similarities between these results and those previously reported for the inactivation of this enzyme by 2'-chloro-2'-deoxyuridine triphosphate suggest a common reaction path for all 2'-halonucleotides. In the pyrimidine series, it was found that 2'-fluoro- and 2'-chloronucleotides partitioned between inactivation and formation of the normal reduction product 2'-deoxynucleotide. Normal reduction predominated with 2'-fluoronucleotides, whereas it was a minor pathway for 2'-chloro-2'-deoxyuridine triphosphate. With 2'-chloro-2'-deoxyuridine triphosphate, the relative partitioning between the two modes was pH dependent: the amount of 2'-deoxyuridine triphosphate formed increased 2.8-fold upon changing from pH 6.1 to pH 8.3. The ability of 2'-arabinohalonucleotides to inactivate ribonucleotide reductase and the variation of partitioning of the pyrimidine analogues with leaving group and reaction pH are consistent with our radical cation hypothesis and support the proposal that the difference between normal catalysis and inactivation is related to the protonation state of the reductase.