Construction, expression and characterization of a chimaeric mammalian-plant aspartic proteinase.
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 degrees C compared with commercial pepsin. Differential scanning calorimetry of the chimaeric enzyme illustrated an approx. 2 degrees C increase in denaturation temperature ( T (m)), with increases in co-operativity and similar enthalpy values. Kinetic analysis indicated an increase in K (m) and decreased k (cat) 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.
Payie
Kenneth G
KG
Department of Food Science, University of Guelph, ON N1G 2W1, Canada.
Tanaka
Takuji
T
Gal
Susannah
S
Yada
Rickey Y
RY
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D016384:Consensus Sequence; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010435:Pepsinogens / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D014908:Triticum / Q000201:enzymology
D007328:Insulin; D010435:Pepsinogens; D011993:Recombinant Fusion Proteins; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A