Conversion of nitrosobenzene to N-phenylacetohydroxamic acid by yeast pyruvate decarboxylase.

In the presence of yeast enzyme concentrate or purified yeast pyruvate decarboxylase, nitrosobenzene was converted in part to N-phenylacetohydroxamic acid. This transformation had to be catalyzed by the enzyme, since the incubation of nitrosobenzene with the cofactor of pyruvate decarboxylase did not produce the hydroxamic acid. Similar incubations conducted with phenylhydroxylamine did not yield any detectable amounts of N-phenylacetohydroxamic acid.

N-phenylglycolhydroxamate production by the action of transketolase on nitrosobenzene.

The incubation of nitrosobenzene with yeast transketolase and D-xylulose 5-phosphate resulted in the production of N-phenylglycolhydroxamic acid. The addition of D-ribose 5-phosphate decreased the amount of hydroxamic acid that was produced. This conversion of nitrosobenzene into the glycollic acid-derived hydroxamic acid was shown to be an enzymic process, and a chemical mechanism for the conversion was proposed.

Peroxide oxidation of indole to oxindole by chloroperoxidase catalysis.

In the presence of chloroperoxidase, indole was oxidized by H2O2 to give oxindole as the major product. Under most conditions oxindole was the only product formed, and under optimal conditions the conversion was quantitative. This reaction displayed maximal activity at pH 4.6, although appreciable activity was observed throughout the entire pH range investigated, namely pH 2.5-6.0. Enzyme saturation by indole could not be demonstrated, up to the limit of indole solubility in the buffer. The oxidation kinetics were first-order with respect to indole up to 8 mM, which was the highest concentration of indole that could be investigated. On the other hand, 2-methylindole was not affected by H2O2 and chloroperoxidase, but was a strong inhibitor of indole oxidation. The isomer 1-methylindole was a poor substrate for chloroperoxidase oxidation, and a weak inhibitor of indole oxidation. These results suggest the possibility that chloroperoxidase oxidation of the carbon atom adjacent to the nitrogen atom in part results from hydrogen-bonding of the substrate N-H group to the enzyme active site.

Synthesis and antibiotic properties of chloramphenicol reduction products.

Analogs of chloramphenicol were prepared for the first time in which the nitro group was replaced by hydroxylamine, nitroso, hydroxamic acid, methyl hydroxamate, and O-acetyl hydroxamate functional groups. These compounds were tested for antibiotic activity in order to determine whether the antibiotic activity of chloramphenicol is mediated by one or more of these potential metabolites of chloramphenicol. None of these analogs was as active as chloramphenicol against the four test organisms, and two of the compounds were essentially devoid of activity. The significance of these findings with regard to the importance of the nitro group to the biological activity of chloramphenicol is discussed.

Chloroperoxidase-catalyzed oxidation of N-methyl-4-chloroaniline.

Chloroperoxidase catalyzed the H2O2 oxidative conversion of N-methyl-4-chloroaniline to 4-chloronitrosobenzene, 4-chloroaniline and a mixture of complex products.

Chloroperoxidase-catalysed oxidation of 4-chloroaniline to 4-chloronitrosobenze.

The incubation of 4-chloroaniline with chloroperoxidase and H2O2 resulted in a rapid formation of 4-chloronitrosobenzene. This enzymic oxidation displayed a pH optimum at 4.4 with a Km of 8.1x10(-4)M and catalytic-centre activity of 312. The initial rate of the reaction was strongly affected by the presence of halide ions. 4-Chlorophenylhydroxylamine was even more rapidly converted into the nitroso compound. A reaction mechanism is proposed on the basis of currently accepted theory for the catalytic action of chloroperoxidae. A noteworthy aspect of this new reaction is the difference in the products previously reported for the action of classical peroxidases on anilines and the single nitroso product resulting from chloroperoxidase oxidation.

The production of hydroxamic acid metabolites of nitrosobenzene by Chlorella pyrenoidosa.

The ability of the green alga Chlorella pyrenoidosa to convert nitrosobenzene (I), phenylhydroxylamine (VI), aniline, and nitrobenzene to hydroxamic acid metabolites was investigated. Only nitrosobenzene and phenylhydroxylamine were partially converted to N-phenylacetohydroxamic acid (Va) and N-phenylglycolhydroxamic acid (Vb), with the latter compound being the major product. The possible mechanisms for the formation of these hydroxamic acid metabolites are discussed. The most plausible explanation for their production is through the interaction of the nitroso group with certain intermediates of thiamine-dependent enzymes. The conversion of phenylhydroxylamine to the hydroxamic acids probably is the result of initial oxidation to nitrosobenzene. Apparently, C. pyrenoidosa lacks nitroreductase and aniline hydroxylase activities, since no metabolic conversions of aniline or nitrobenzene were observed. The potential environmental significance of hydroxamic acid production from nitrosoaromatics is discussed.

The action of chloride peroxidase on 4-chloroaniline. N-oxidation and ring halogenation.

Chloride peroxidase catalyses both the ring halogenation and N-oxidation reactions of 4-chloroaniline by H2O2 and either KCl or KBr. In the absence of any halide salt only the N-oxidation reaction was observed, with the resulting conversion of 4-chloroaniline into 4-chloronitrosobenzene. The N-oxidation reaction proceeded even more rapidly in the presence of Cl- or Br-, in spite of the fact that ring halogenation was also a rapid reaction. The enhancement of N-oxidation was highly dependent on the pH of the media and displayed an optimum in the region of pH 3.5-4.0. No rate enhancement was observed above pH 5.5. KF partially inhibited the rate of N-oxidation in a pH-dependent manner. On the basis of calculated catalytic-centre activity the N-oxidation reaction was the major reaction at pH 3.5 or higher, whereas the ring-halogenation reaction became the major reaction below pH 3.5. In the presence of high concentrations of 4-chloroaniline relative to H2O2 the reaction intermediate, 4-chlorophenylhydroxylamine, was detected for the first time in a chloride peroxidase-catalysed reaction with this arylamine substrate. These findings were interpreted on the basis of current knowledge concerning the mechanism of action of chloride peroxidase.