Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from <i>Araujia angustifolia</i> latex

Abstract

Araujiain aII, the protease with highest specific activity purified from latex of <i>Araujia angustifolia</i> (Apocynaceae), shows optimum proteolytic activity at alkaline pH, and it is completely inhibited by the irreversible inhibitor of cysteine proteases <i>trans</i>-epoxysucciny-l-leucyl-amido(4-guanidino) butane. It exhibits esterolytic activity on several N-α-Cbz-amino acid p-nitrophenyl esters with a preference for Gln, Ala, and Gly derivatives. Kinetic enzymatic assays were performed with the thiol proteinase substrate p-Glu-Phe-Leu-p-nitroanilide (Kₘ = 0.18 ± 0.03 mM, k_cat = 1.078 ± 0.055 s⁻¹, k_cat/Kₘ = 5.99 ± 0.57 s⁻¹ mM⁻¹). The enzyme has a pI value above 9.3 and a molecular mass of 23.528 kDa determined by mass spectrometry. cDNA of the peptidase was obtained by reverse transcription-PCR starting from total RNA isolated from latex. The deduced amino acid sequence was confirmed by peptide mass fingerprinting analysis. The N-terminus of the mature protein was determined by automated sequencing using Edman’s degradation and compared with the sequence deduced from cDNA. The full araujiain aII sequence was thus obtained with a total of 213 amino acid residues. The peptidase, as well as other Apocynaceae latex peptidases, is a member of the subfamily C1A of cysteine proteases. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22–Cys63 and Cys56–Cys95) in the alpha domain, and another one (Cys150–Cys201) in the beta domain, as was suggested by molecular modeling.