Purification, subunit structure, and partial amino acid sequence of metapyrocatechase.

Abstract

Crystalline metapyrocatechase with a specific activity of about 320 pmol/min/mg of protein was obtained from an extract of Pseudomonasputida mt-2 grown in a medium containing meat extract, peptone, and benzoate.This preparation was about 3 times more active and contained 3 times more iron (3.4 g atoms/mol) than the preparation reported previously (Nozaki, M., Kagamiyama, H., and Hayaishi, 0. (1963) Biochem 2. 338, 582-590), although these preparations had the same molecular weight (140,000).The variation of specific activity of the enzyme was found to correlate with the iron content; the fully active preparations appeared to contain 4 g atoms of iron/mol of enzyme.The enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M, = 35,000.The NH2-terminal sequence starting with methionine was determined up to 53 residues by Edman degradation.During the degradation, a single amino acid was released at each step.The COOH-terminal sequence (6 residues) starting with threonine was determined by carboxypeptidase A digestion.These results indicate that the enzyme consists of four identical subunits.Each subunit appears to contain 1 g atom of iron essential for the activity.Results of the partial sequence analyses also provided us with useful information for locating the structural gene of metapyrocatechase on the DNA fragment as will be described in the following paper (Nakai, C., Kagami-