Biochemical comparison of two proteolytic enzymes from Carica candamarcensis: structural motifs underlying resistance to cystatin inhibition.
The lattices of Carica candamarcensis and Carica papaya, members of the Caricaceae family, contain isoforms of cysteine proteinases that help protect these plants against injury. In a prior study, we fractionated 14 discrete proteinaceous components from C. candamarcensis, two of them displaying mitogenic activity in mammalian cells. In this study, we compared the kinetic parameters of one of the mitogenic proteinases (CMS2MS2) with one of the isoforms displaying the highest enzyme activity of this group (CMS1MS2). Both enzymes display a similar Km value with either BAPNA (Benzoyl-Arg-pNA) or PFLPNA (Pyr-Phe-Leu-pNA), but the kcat of CMS1MS2 is about 14-fold higher for BAPNA and 129-fold higher with PFLPNA. While both enzymes are inhibited by E-64 and iodoacetamide, chicken cystatin fully inhibits CMS1MS2, but scarcely affects activity of CMS2MS2. Based on the structure of these proteins and other enzymes from the Caricaceae family whose structures have been resolved, it is proposed that Arg(180) located in the cleft at the active site in CMS2MS2 is responsible for its resistance to cystatin.
Gomes
Marco Túlio R
MT
Departamento de Bioquímica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Ribeiro
Henrique A
HA
Lopes
Miriam T P
MT
Guzman
Fanny
F
Salas
Carlos E
CE
D000818:Animals; D031241:Caricaceae / Q000201:enzymology*; D002645:Chickens; D015891:Cystatins; D057056:Cysteine Proteases / Q000737:chemistry* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors; D007460:Iodoacetamide; D008934:Mitogens / Q000737:chemistry* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D020033:Protein Isoforms; D013329:Structure-Activity Relationship
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D008934:Mitogens; D010940:Plant Proteins; D020033:Protein Isoforms; D057056:Cysteine Proteases; D010447:Peptide Hydrolases; D007460:Iodoacetamide