Mutagenesis and kinetic studies of a plant cysteine proteinase with an unusual arrangement of acidic amino acids in and around the active site

We report the cloning, overexpression, kinetic analysis, and modeling of th e tertiary structure of an unusual plant cysteine proteinase. Ananain (EC 3 .4.22.31), from Ananas comosus (pineapple) is distinguished from all other cysteine proteinases in the papain superfamily by having a unique combinati on of acidic amino acids. As well as lacking the acidic residue immediately preceding the active site histidine (position 158 in papain), it also lack s the extensive surface network of acidic residues that were postulated to compensate for the loss of charge at position 158 in mammalian cathepsins. Ananain has the fewest acidic residues, so far reported, of any plant cyste ine proteinase, but two of the carboxyl residues (E50 and E35) postulated t o have an enabling role in catalysis, the so-called "electrostatic switch", remain conserved. Comparisons of the kinetics of recombinant wild-type ana nain with E50A and E35A mutants proves that these charged groups are not es sential for catalysis. Hence this research does not confirm the presence of an electrostatic switch in this cysteine proteinase, and the role of acidi c residues in the enhancement of catalytic competence in these enzymes is d iscussed in light of this new evidence.