Lidocaine metabolism by rabbit-liver homogenate and detection of a new metabolite.

Metabolism of lidocaine in rabbit liver 9000 g supernatant fraction was examined. A capillary g.l.c. assay was developed to separate seven known metabolites of lidocaine, and all seven metabolites were identified in extracts of incubations of lidocaine with rabbit-liver fractions. These metabolites were monoethylglycinexylidide(I), glycinexylidide(II), 3-hydroxymonoethylglycinexylidide(III), 3-hydroxylidocaine(IV), 4-hydroxylidocaine(V), xylidine(VI) and 4-hydroxyxylidine(VII). A new metabolite, 2-amino-3-methylbenzoic acid(VIII), was identified in extracts of incubations of lidocaine with rabbit-liver fractions, by comparison of the mass-spectral fragmentation patterns and g.l.c. retention time with those of the authentic compound. The formation of VIII is dependent on protein, NADPH, time, O2, and the presence of soluble enzymes. Quantitative analysis of metabolites I-VIII after a two hour incubation accounts for 89% of the metabolized lidocaine.

p-Aminobenzoate-p-aminobenzoate.

p-Aminobenzoate (PABA) synthase from Bacillus subtilis is an aggregate composed of two nonidentical subunits and has the following properties. (i) In crude extracts this enzyme catalyzes the formation of PABA in the presence of chorismate and either glutamine (amidotransferase) or ammonia (aminase). The amidotransferase activity is about 5- to 10-fold higher than the aminase activity and is stable for at least 1 week when frozen at -70 C. (II) Although no divalent cation requirement could be demonstrated with crude extracts, 2 mM ethylene-diaminetetraacetic acid completely inhibits both activities. (iii) After ammonium sulfate fractionation both the aminase and amidotransferase activities require Mg2+ and guanosine in addition to the substrates indicated above for optimal activity. The guanosine requirement can be replaced by guanosine 5'-monophosphate, guanosine 5'-diphosphate, and guanosine 5'-triphosphate but not by guanine, adenosine 5'-triphosphate, uridine 5'-triphosphate, cytidine 5'-triphosphate, thymidine 5'-triphosphate, inorganic phosphate, and phosphoribosylpyrophosphate. Furthermore, at a pH above 7.4 or below 6.4 activity is rapidly lost a 4 C, or -60 C. (IV) The enzyme is composed of two non-identical subunits, designated subunit A and subunit X. Subunit A has an estimated molecular weight of 31,000, whereas subunit X has an estimated molecular weight of 19,000. Subunit A has aminase activity but no amidotransferase activity; a mutation at the pabA locus results in the loss of PABA synthase activity. Subunit X, which is also a component of the anthranilate synthase complex, has no PABA synthase activity itself but complexes with subunit A to give an AX aggregate that can use glutamine as a substrate. (v) The molecular weight of the AX complex has been estimated at 50,000, suggesting a 1:1 ratio of subunits. (vi) The enzyme is readily associated and dissociated.

4-Nitrobenzoic acid reductase of Ascaris lumbricoides var suum. Substrate specificity and reaction products.

1. The substrate specificity of nitro-reductase from Ascaris lumbricoides varsum was determined. This enzyme reduced nitrobenzene, 4-nitrohippuric acid and the isomers of nitrophenol, nitroanisole, nitrobenzoic acid, nitrobenzaldehyde and nitrobenzyl alcohol. The same enzyme preparation reduced azobenzene, 4-dimethylaminoazobenzene and 1,2-dimethyl-4-(4-carboxyphenylazo)-5-hydroxybenzene. Nitrobenzaldehyde isomers were not reduced to the alcohols. 2. The products of nitro- and azo-reduction were the corresponding amines, no hydroxylamino or hydrazo compounds were detected. 3. The pH optima and cofactor requirements were the same for both azo- and nitro-reduction and neither reaction was inhibited by oxygen. 4. Ammonium sulphate fractionation failed to separate azo- and nitro-reductase activities. The molecular weight of both azo- and nitro-reductase was about 130 000.

Biosynthesis of 4-aminobenzoate in Escherichia coli.

Two different mutations (pabA and pabB) affecting 4-aminobenzoate biosynthesis were obtained in strains of Escherichia coli lacking chorismate mutase and anthranilate synthetase activity, thus allowing study of the pathway of biosynthesis of 4-aminobenzoate by use of cell extracts of strains carrying the pab mutations. Two components with approximate molecular weights of 9,000 (component A) and 48,000 (component B) are concerned in the biosynthesis of 4-aminobenzoate from chorismate by E. coli. No diffusible intermediate compound could be detected.