Construction, expression and characterization of a chimaeric mammalian-plant aspartic proteinase

Kenneth G. Payie, Takuji Tanaka, Susannah Gal, Rickey Y. Yada

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Aspartic proteinases are a well-characterized class of proteinases. In plants, all nascent aspartic proteinases possess a 100-amino-acid, plant-specific sequence (PSS) within their C-terminal lobe, presumed to possess a targeting role in vivo. In this study, the PSS domain from the Arabidopsis thaliana aspartic proteinase was inserted into porcine pepsinogen at the identical location found in nascent plant aspartic proteinases, to create a chimaeric mammalian-plant enzyme. Based on enzymic activity, this chimaeric enzyme demonstrated increases in pH stability above 6 and temperature stability above 60°C compared with commercial pepsin. Differential scanning calorimetry of the chimaeric enzyme illustrated an approx. 2°C increase in denaturation temperature (Tm), with increases in co-operativity and similar enthalpy values. Kinetic analysis indicated an increase in Km and decreased kcat compared with pepsin, but with a catalytic efficiency similar to the monomeric plant aspartic proteinase from wheat. Using oxidized insulin B-chain, the chimaeric enzyme demonstrated more restricted substrate specificity in comparison with commercial pepsin. This study highlights the use of a chimaeric enzyme strategy in order to characterize unique protein domains within enzyme families, and, for the first time, a putative structure-function role for the PSS as it pertains to plant aspartic proteinases.

Original languageEnglish (US)
Pages (from-to)671-678
Number of pages8
JournalBiochemical Journal
Volume372
Issue number3
DOIs
StatePublished - Jun 15 2003

Fingerprint

Aspartic Acid Proteases
Pepsin A
Enzymes
Pepsinogen A
Denaturation
Temperature
Differential Scanning Calorimetry
Substrate Specificity
Differential scanning calorimetry
Enthalpy
Arabidopsis
Peptide Hydrolases
Triticum
Insulin
Swine
Amino Acids
Kinetics
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Payie, Kenneth G. ; Tanaka, Takuji ; Gal, Susannah ; Yada, Rickey Y. / Construction, expression and characterization of a chimaeric mammalian-plant aspartic proteinase. In: Biochemical Journal. 2003 ; Vol. 372, No. 3. pp. 671-678.
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Construction, expression and characterization of a chimaeric mammalian-plant aspartic proteinase. / Payie, Kenneth G.; Tanaka, Takuji; Gal, Susannah; Yada, Rickey Y.

In: Biochemical Journal, Vol. 372, No. 3, 15.06.2003, p. 671-678.

Research output: Contribution to journalArticle

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