Studies on the interaction of p-hydroxybenzoate hydroxylase with NADPH. Effects of pH and substrates on the enzyme . NADPH complex formation.

H Shoun,N Higashi,T Beppu,S Nakamura,K Hiromi,K Arima

doi:10.1016/s0021-9258(19)86615-2

Abstract

Studies on the interaction of p-hydroxybenzoate hydroxylase with NADPH. Effects of pH and substrates on the enzyme . NADPH complex formation.

Highlights

Faculty of Agriculture, The University of Tokyo, Yayoi, Bunkyo-ku, Kitasato University School of Medicine, Asamizodai, Sagamihara, Science and Technology, Faculty of Agriculture, Kyoto University, Kei
At least two spectral intermediates were observed during anaerobic reduction of oxidized p-hydroxybenzoate hydroxylase by NADPH in the presence of phydroxybenzoate
We describe results of studies on the interaction of p-hydroxybenzoate hydroxylase from P. desmolytica with NADPH by spectrophotometric measurements and kinetic analyses

Summary

PROCEDURES

IAM 1123 as previously reported [17]. Purified enzyme stored as the crystalline of the enzyme.substrate complex [17] in an ammonium sulfate solution (pH 6.0) was exposed to both dialysis and gel filtration before use to exclude the substrate completely. Process of the complex formation between p-hydroxybenzoate hydroxylase and NADPH was observed by the RA 401 stoppedflow apparatus with accumulation device. The upper space of each reservoir syringe of the apparatus was kept anaerobic under continuous flow of nitrogen gas. The upper space was substituted by nitrogen gas, and the enzyme solution in the cuvette and NADPH solution in the side arm were kept anaerobic by the glucose oxidase-catalase system. Both solutions were mixed after the anaerobic condition was attained, and the decrease in absorbance at 450 nm due to bound FAD was recorded at 25°C with the spectrophotometer. The buffer solution used was 50 mrvr potassium-phosphate buffer (with various pH values), unless otherwise stated

RESULTS

NADPHI

I I I 1I I

DISCUSSION