Structural and biological characterization of a novel acutobin-like enzyme isolated from the venom of the sharp-nosed pit viper (Deinagkistrodon acutus).
We previously reported the purification of a serine proteinase from the venom of the sharp-nosed pit viper (Deinagkistrodon acutus) using a combination of affinity chromatography and ion-exchange chromatography [Xin, Dong, Wang and Li, R. (2007) J. Chromatogr. B 859, 111-118]. The high fibrinogen-clotting activity [2025 NIH (National Institutes of Health) units/mg] of this protein indicated that it may have great potential as a drug for treating thrombolysis. In order to systemically determine the purified protein's structure and activity, it was characterized using the following methods: MS, isoelectric focusing, deglycosylation analysis, amino acid composition analysis, peptide mass fingerprinting, N-terminal amino acid sequencing, CD, hydrophobic-site analysis and bioactivity assays. In addition, a fluorescence probe was synthesized and conjugated to the protein in order to analyse its active site. The results indicated that the protein is a novel acutobin-like enzyme (designated acutobin II) with strong clotting and esterase activities and is composed of a 28 kDa peptide chain plus approx. 6 kDa of O-linked glycan chains. The protein contains 249 amino acids and, remarkably, no tryptophan residues. The pI of the protein is 4.8+/-0.2. The protein's secondary structure is dominated by beta-sheets (49%) and random coils (43%), and its tertiary structure does not contain any metal ions or disulfide bonds and possesses only one hydrophobic pocket. Analysis revealed that the hydrophobic pocket is most likely the enzymatic active site.
Xin
Yu
Y
Ministry of Education Key Laboratory of Microbial Metabolism, College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.
Dong
Dexian
D
Chen
Dezhao
D
Li
Rongxiu
R
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D003435:Crotalid Venoms / Q000737:chemistry*; D004795:Enzyme Stability; D004798:Enzymes / Q000737:chemistry* / Q000378:metabolism*; D004950:Esterases / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D013917:Thrombin / Q000737:chemistry*; D014757:Viper Venoms / Q000201:enzymology*; D017819:Viperidae
D003435:Crotalid Venoms; D004798:Enzymes; D014757:Viper Venoms; D005340:Fibrinogen; D004950:Esterases; D013917:Thrombin; C508893:acutobin, Deinagkistrodon acutus; D000581:Amidohydrolases; C019665:amidase