Functional chimera of porcine pepsin prosegment and Plasmodium falciparum plasmepsin II.
Proplasmepsin II (zPMII) represents a unique member of the aspartic proteinase family, with a prosegment-enzyme interaction that is thus far unique among the pepsin-like proteases. The role of the prosegment in aspartic proteinase structure and function was investigated by generating two chimeric proteins, one with the pepsinogen prosegment fused to the mature region of PMII (pepproPMII) and a second with the prosegment of PMII fused to pepsin (PMIIpropep). Both chimeras were expressed using Escherichia coli; however, PMIIpropep was extremely unstable suggesting protein misfolding. Alternatively, pepproPMII was capable of both autoactivation and hydrolysis of a synthetic substrate. Similarly, when the PMII enzyme was expressed without a prosegment, it too exhibited activity against the synthetic enzyme. CD measurements indicated that pepproPMII had reduced thermal stability when compared with zPMII. This reduction of temperature stability may have resulted from the inability of the pepsinogen prosegment to stabilize the C-terminal domain of the PMII enzyme. The ability of PMII to fold in the presence of a completely non-homologous prosegment and in its absence suggests that prosegment is not critical to obtaining a functional enzyme in all pepsin-like enzymes but likely plays a role in protein stabilization.
Parr-Vasquez
Charity L
CL
Department of Food Science, University of Guelph, Guelph, ON, Canada N1G2W1.
Yada
Rickey Y
RY
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D010434:Pepsin A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology; D015202:Protein Engineering; D017510:Protein Folding; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013552:Swine
D004792:Enzyme Precursors; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A; C100637:plasmepsin II