The N-terminal propeptide of Vibrio vulnificus extracellular metalloprotease is both an inhibitor of and a substrate for the enzyme.
Vibrio vulnificus, a marine bacterium capable of causing wound infection and septicemia, secretes a 45-kDa metalloprotease (vEP) with many biological activities. The precursor of vEP consists of four regions: a signal peptide, an N-terminal propeptide (nPP), a C-terminal propeptide, and the mature protease. Two forms of vEP-vEP-45, which contains the mature protease plus the C-terminal propeptide, and vEP-34, which contains only the mature protease-were expressed in Escherichia coli and purified. vEP-45 and vEP-34 had similar activities with azocasein as a substrate, but vEP-34 had reduced activity toward insoluble proteins. The nPP of vEP was expressed as a His tag fusion protein, and its effect on vEP activity was investigated. nPP inhibited the activities of both vEP-45 and vEP-34 but not that of thermolysin, a different but related zinc-dependent protease. The inhibition of vEP by nPP was further examined using vEP-34 as a representative enzyme. The inhibition could be completely reversed under conditions of low enzyme and propeptide concentrations and with prolonged incubation, which resulted from the degradation of nPP by vEP. However, even at high nPP and vEP concentrations, inhibition of vEP by nPP at high temperatures was not effective, resulting in the degradation of both nPP and vEP. These results demonstrate that the nPP of vEP could bind to vEP and inhibit its activity, resulting in the degradation of the propeptide.
Chang
Alan K
AK
Research Center for Proteineous Materials, Chosun University, 375 Seosuk-dong Dong-gu, Gwangju 501-759, Republic of Korea.
Park
Jong Woo
JW
Lee
Eun Hee
EH
Lee
Jung Sup
JS
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D013379:Substrate Specificity; D013997:Time Factors; D041261:Vibrio vulnificus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D045726:Metalloproteases