4-alkyl-o-quinone/2-hydroxy-p-quinone methide isomerase from the larval hemolymph of Sarcophaga bullata. I. Purification and characterization of enzyme-catalyzed reaction

• Published in: The Journal of biological chemistry 1990-10, Vol.265 (28), p.16992-16999
• Main Authors: Saul, S J, Sugumaran, M
• Format: Article
• Language: English
• Subjects: 4-alkyl-o-quinone isomerase; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; CHEMICOPHYSICAL PROPERTIES; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Diptera; Enzymes and enzyme inhibitors; ENZYMIC ACTIVITY; Fundamental and applied biological sciences. Psychology; HAEMOLYMPH; HEMOLINFA; Hemolymph - enzymology; HEMOLYMPHE; Insecta - enzymology; Intramolecular Oxidoreductases; ISOLATION; ISOMERASAS; ISOMERASE; ISOMERASES; Isomerases - isolation & purification; Isomerases - metabolism; Kinetics; Larva; LARVAE; LARVAS; LARVE; Magnetic Resonance Spectroscopy; MOLECULAR WEIGHT; NEOBELLIERIA BULLATA; PESO MOLECULAR; POIDS MOLECULAIRE; PROPIEDADES FISICO-QUIMICAS; PROPRIETE PHYSICOCHIMIQUE; QUINONAS; QUINONE; QUINONES; Sarcophagidae; SCLEROTIZATION; Spectrophotometry; Substrate Specificity; Thermodynamics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)44858-7

An enzyme which catalyzes the conversion of certain 4-alkyl-o-benzoquinones to 2-hydroxy-p-quinone methides has been purified to apparent homogeneity from the hemolymph of Sarcophaga bullata by employing conventional protein purification techniques. The purified enzyme migrated with an approximate molecular weight of 98,000 on gel filtration chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it migrated as a single band with a molecular weight of 46,000, indicating that it is made up of two identical subunits. It exhibited a pH optimum of 6.0 and readily converted chemically synthesized as well as enzymatically generated quinones derived from N-acetyldopamine, N-beta-alanyldopamine, and 3,4-dihydroxyphenethyl alcohol to highly unstable 2-hydroxy-p-quinone methides. The quinone methides thus formed were rapidly and nonenzymatically hydrated to form side chain hydroxylated o-diphenols as the stable product. In support of this proposition, when the enzyme reaction with N-acetyldopamine quinone was conducted in the presence of 10% methanol, racemic beta-methoxy-N-acetyldopamine was recovered as an additional product. The quinones of N-acetylnorepinephrine, N-beta-alanylnorepinephrine, and 3,4-dihydroxyphenylglycol were also attacked by the isomerase, resulting in the formation of N-acetylarterenone, N-beta-alanylarterenone and 2-hydroxy-3‘,4‘-dihydroxyacetophenone, respectively as the stable products. The isomerase converted the dihydrocaffeiyl methyl amide quinone to its quinone methide analog which rapidly tautomerized to yield caffeiyl methyl amide. The importance of quinone isomerase in insect immunity and sclerotization of insect cuticle is discussed.