Isolation and characterization of a small catalytic domain released from the adenylate cyclase from Escherichia coli by digestion with trypsin

M. M. Holland, T. K. Leib, J. A. Gerlt

Research output: Contribution to journalArticle

Abstract

An expression plasmid containing a hybrid gene encoding a protein having the primary amino acid sequence of the adenylate cyclase from Escherichia coli was constructed. When the gene was induced, the adenylate cyclase could be expressed at high levels in a cya- strain of E. coli. The majority of the enzymatic activity and protein (having a molecular weight of 95,000) induced was insoluble. However, treatment of the insoluble fraction of cell lysates with trypsin resulted in both an increase in and solubilization of the total amount of adenylate cyclase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the soluble protein produced by treatment with trypsin revealed a polypeptide having a molecular weight of 30,000. This soluble, catalytically active fragment of adenylate cyclase was purified and subjected to amino-terminal sequence analyses; two amino-terminal sequences were identified beginning at residue 82 and at residue 342 of the intact enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified fragment followed by either silver or Coomassie Blue staining revealed the presence of only a single polypeptide having a molecular weight of 30,000; a short oligopeptide associated with the amino terminus at residue 342 could not be detected. Site-directed mutagenesis was used to place a stop codon at residue 341; the truncated enzyme was catalytically active, so the short oligopeptide is not necessary for catalysis. The K(m) for ATP, the K(a) for Mg2+, and the V(max) determined for the product containing the 30,000-dalton fragment were similar to the values reported for the intact enzyme from E. coli.

Original languageEnglish (US)
Pages (from-to)14661-14668
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number29
StatePublished - 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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