Domain organization and a protease-sensitive loop in eukaryotic ornithine decarboxylase

Andrei L. Osterman, Deirdre V. Lueder, Mary Quick, David Myers, Bertram J. Canagarajah, Margaret A. Phillips

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Trypanosoma brucei ornithine decarboxylase was reconstituted by coexpression of two polypeptides corresponding to residues 1-305 and residues 306-425 in Escherichia coli. The two peptides were coexpressed, at wild-type levels, from a single transcriptional unit that was separated by a 15-nucleotide untranslated region containing a ribosome binding site. The fragmented enzyme was purified and analyzed. The N- and C-terminal peptides are tightly associated into a fully active tetramer which has the same molecular weight as the native dimer. The kinetic constants (Km and kcat) measured for the decarboxylation of ornithine are identical to those obtained for the wild-type enzyme. These results suggest that the enzyme is organized into two structural domains, with a domain boundary in the region of amino acid 305. In contrast, the individual N- and C-terminal peptides are expressed primarily as inclusion bodies. Small quantities of soluble N-terminal peptide could be purified. This truncated protein is capable of inhibiting the wild-type enzyme, suggesting that it is folded into a native-like structure. Limited proteolysis with trypsin or chymotrypsin identifies a likely surface loop at amino acids 160-170, present in both the mouse and T. brucei enzyme, which positions one or more functionally important active site residues (e.g., Lys 169). Kinetic analysis of a chimeric enzyme composed of T. brucei and mouse ornithine decarboxylase suggests that the substrate carboxylate binding determinant is located between residues 1 and 170.

Original languageEnglish (US)
Pages (from-to)13431-13436
Number of pages6
JournalBiochemistry
Volume34
Issue number41
StatePublished - 1995

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Ornithine Decarboxylase
Peptide Hydrolases
Trypanosoma brucei brucei
Enzymes
Peptides
Proteolysis
Untranslated Regions
Amino Acids
Decarboxylation
Kinetics
Ornithine
Inclusion Bodies
Chymotrypsin
Ribosomes
Dimers
Trypsin
Escherichia coli
Catalytic Domain
Nucleotides
Molecular Weight

ASJC Scopus subject areas

  • Biochemistry

Cite this

Osterman, A. L., Lueder, D. V., Quick, M., Myers, D., Canagarajah, B. J., & Phillips, M. A. (1995). Domain organization and a protease-sensitive loop in eukaryotic ornithine decarboxylase. Biochemistry, 34(41), 13431-13436.

Domain organization and a protease-sensitive loop in eukaryotic ornithine decarboxylase. / Osterman, Andrei L.; Lueder, Deirdre V.; Quick, Mary; Myers, David; Canagarajah, Bertram J.; Phillips, Margaret A.

In: Biochemistry, Vol. 34, No. 41, 1995, p. 13431-13436.

Research output: Contribution to journalArticle

Osterman, AL, Lueder, DV, Quick, M, Myers, D, Canagarajah, BJ & Phillips, MA 1995, 'Domain organization and a protease-sensitive loop in eukaryotic ornithine decarboxylase', Biochemistry, vol. 34, no. 41, pp. 13431-13436.
Osterman AL, Lueder DV, Quick M, Myers D, Canagarajah BJ, Phillips MA. Domain organization and a protease-sensitive loop in eukaryotic ornithine decarboxylase. Biochemistry. 1995;34(41):13431-13436.
Osterman, Andrei L. ; Lueder, Deirdre V. ; Quick, Mary ; Myers, David ; Canagarajah, Bertram J. ; Phillips, Margaret A. / Domain organization and a protease-sensitive loop in eukaryotic ornithine decarboxylase. In: Biochemistry. 1995 ; Vol. 34, No. 41. pp. 13431-13436.
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