Functional analysis of the cucumisin propeptide as a potent inhibitor of its mature enzyme.
Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH(2)-terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a K(i) value of 6.2 ± 0.55 nm. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. Deletion analysis clearly showed that two hydrophobic regions, Asn(32)-Met(38) and Gly(97)-Leu(103), in the cucumisin propeptide were important for its inhibitory activity. Site-directed mutagenesis also confirmed the role of a Val(36)-centerd hydrophobic cluster within the Asn(32)-Met(38) region in cucumisin inhibition. Circular dichroism spectroscopy revealed that the cucumisin propeptide had a secondary structure without a cognate protease domain and that the thermal unfolding of the propeptide at 90 °C was only partial and reversible. A tripeptide, Ile(35)-Val(36)-Tyr(37), in the Asn(32)-Met(38) region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease.
Nakagawa
Masataka
M
Laboratory of Biochemistry, Graduate School of Agricultural Science, Kobe University, Nada, Kobe 657-8501, Japan.
Ueyama
Megumi
M
Tsuruta
Hiroki
H
Uno
Tomohide
T
Kanamaru
Kengo
K
Mikami
Bunzo
B
Yamagata
Hiroshi
H
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019662:Cucurbitaceae / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000502:physiology; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000201:enzymology / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D013381:Subtilisins; C095313:ara12 protein, Arabidopsis; C041541:cucumisin