Soluble artificial metalloproteases with broad substrate selectivity, high reactivity, and high thermal and chemical stabilities.
To design soluble artificial proteases that cleave peptide backbones of a wide range of proteins with high reactivity, artificial active sites comprising the Cu(II) complex of 1-oxa-4,7,10-triazacyclodedecane (oxacyclen) and the aldehyde group were synthesized. The aldehyde group was employed as the binding site in view of its ability to reversibly form imine bonds with ammonium groups exposed on the surfaces of proteins, and Cu(II) oxacyclen was exploited as the catalytic group for peptide hydrolysis. The artificial metalloproteases synthesized in the present study cleaved all of the protein substrates examined (albumin, gamma-globulin, myoglobin, and lysozyme). In addition, the activity of the best soluble artificial protease was enhanced by up to 190-fold in terms of kcat/Km. When the temperature was raised to 80 degrees C, the activities of the artificial proteases were significantly enhanced. The activity of the artificial protease was not greatly affected by surfactants, including sodium dodecyl sulfate. The intermediacy of the imine complex formed between the artificial protease and the protein substrate was supported by an experiment using sodium cyanoborohydride. Soluble artificial metalloproteases with broad substrate selectivity, high reactivity, high thermal and chemical stabilities, and small molecular weights were thus synthesized by positioning the aldehyde group in proximity to Cu(II) oxacyclen.
Kim
Min Gyum
MG
Department of Chemistry, Seoul National University, Seoul, 151-747, Korea.
Yoo
Sang Ho
SH
Chei
Woo Suk
WS
Lee
Tae Yeon
TY
Kim
Hye Mi
HM
Suh
Junghun
J
D002384:Catalysis; D000080027:Cyclams; D004795:Enzyme Stability; D006571:Heterocyclic Compounds / Q000737:chemistry; D045726:Metalloproteases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature
D000080027:Cyclams; D006571:Heterocyclic Compounds; D008670:Metals; C038072:cyclen; D045726:Metalloproteases