Mutagenesis and Kinetic Studies of a Plant Cysteine Proteinase with an Unusual Arrangement of Acidic Amino Acids in and around the Active Site^,

Abstract

We report the cloning, overexpression, kinetic analysis, and modeling of the 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 combination of acidic amino acids. As well as lacking the acidic residue immediately preceding the active site histidine (position 158 in papain), it also lacks 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 cysteine proteinase, but two of the carboxyl residues (E50 and E35) postulated to have an enabling role in catalysis, the so-called "electrostatic switch", remain conserved. Comparisons of the kinetics of recombinant wild-type ananain with E50A and E35A mutants proves that these charged groups are not essential for catalysis. Hence this research does not confirm the presence of an electrostatic switch in this cysteine proteinase, and the role of acidic residues in the enhancement of catalytic competence in these enzymes is discussed in light of this new evidence.

Electrostatic properties in the catalytic site of papain: A possible regulatory mechanism for the reactivity of the ion pair

“…We found the Asp57 residue as the most promising candidate for an essential ionizable residue in contrast with the work of Pinitglang et al, 23 who found Glu50 and Glu35 as the most promising ones. Site-directed mutagenesis results involving a Glu50/Ala mutation in caricain 42 and Glu35/ Ala and Glu50/Ala mutations in ananain 43 practically discarded these residues as essential for catalytic competence (at least in these specific enzymes).…”

“…The 17 molecules selected correspond to the following molecules from the 9pap PDB file: 13,16,28,29,40,42,43,97,100,102,135,159,191,203,207,214, and 216. The water oxygen atoms after the refinement step show an average distance of 0.46 Å from their original position in the PDB file.…”

“…The water oxygen atoms after the refinement step show an average distance of 0.46 Å from their original position in the PDB file. The six water molecules (16,42,43,102,159, and 203) with at least one atom with a distance Յ 4 Å from the (Cys25)-HG and (His159)-ND1 atoms had their individual electrostatic contribution calculated using the exact ab initio procedure (Hartree-Fock/6-31G*). The remaining water molecules had their individual electrostatic contribution computed using the multicentered multipolar expansion method.…”

“…However, the precise mechanism of this electrostatic switch, the acidic residues that modulate the ion pair catalytic competence and whether this mechanism is present in other cysteine proteinases, remain to be determined. These are important topics of research that in the past few years have been the subject of protein engineering studies involving plant cysteine proteinases [42][43][44] and chemically mutated Ser3 Cys serine proteinases. 45 In this article, we investigated the electrostatic profile in the Cys25/His159 catalytic region of papain, aiming to identify its characteristics and the possible enzymatic regulatory mechanisms in the region where the catalytic residues interact with the ligand and with the active site water molecules.…”

Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme

“…Recently, however, evidence has accumulated that this ion-pair can exist at pH values at which the enzyme is not active [15][16][17][18] and that deprotonation of another site on the enzyme is required for papain-like enzymes to become catalytically competent. Several groups have investigated, by site-directed mutagenesis, residues in papain and related enzymes such as caricain (another proteinase from papaya) and ananain [from Ananas comosus (pineapple)] in an attempt to understand the importance of the electrostatic environment to catalysis and to pinpoint single residues which can confer catalytic competence [18][19][20][21]. To date, however, such residues have not been un-…”