Crystal structure of a caricain D158E mutant in complex with E‐64

Abstract

'similar' hydrogen bonds to the Asp but the Glu could not. Abstract The structure of the D158E mutant of caricain (previously known as papaya protease omega) in complex withThese authors clearly consider that Asn could fit neatly into E-64 has been determined at 2.0 A resolution (overall R factor the unperturbed structure whilst the Glu side chain is too long 19.3%). The structure reveals that the substituted glutamate to fit. Taylor et al. [19] report the effect of substituting Ala, makes the same pattern of hydrogen bonds as the aspartate in Asn and Glu for Asp-158 in caricain and highlight the potennative caricain. This was not anticipated since in the native tial multiple ionizations involved in activity. The kcJKm valstructure there is insufficient room to accommodate the ues for the caricain mutants rank Asp > Glu >Asn > Ala. glutamate side chain. The glutamate is accommodated in the This suggests that for caricain the Glu-158 mutant is the mutant by a local expansion of the structure demonstrating that most similar in structure as it has the highest activity. Clearly, small structural changes are responsible for the change in the same hydrogen bonds could be made by the glutamate if it activity, could be accommodated within the structure.To establish the position of the glutamate side chain at Key words: Kinetically influential mutant; Cysteine protease; Site directed mutagenesis; X-ray crystallography; position 158 in caricain we have determined the structure by Enzyme-inhibitor complex X-ray crystallography. We discuss this structure and activity, and the implications for understanding the effect of mutations at the corresponding position in papain.

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

“…8A and Table 3). The arrangement is highly similar to the binding mode found in papain and other cysteine proteases of the same family and causes only a minor expansion of the active site to accommodate the proper coordinate chemistry (60,61). The oxyanion hole is occupied by the carboxylic acid group of E-64, and the leucyl moiety is located in the S 2 pocket of dionain-1, where it is held in place by hydrophobic interactions and by backbone hydrogen bonding of the succeeding E-64 amide nitrogen to the carbonyl oxygen of Gly 69 (Fig.…”

“…The Z-FR-AMC substrate was used for kinetic profiling either at 0.25 mM or in a dilution series to determine kinetic constants. The catalytic competence of dionain-1 at different temperatures (4,20,30,40,50,60,70, and 80°C) was probed by end point fluorescence after 15-min incubation of dionain-1 in preheated 50 mM sodium acetate, pH 5.5, assay buffer containing 2 mM DTT and 0.25 mM Z-FR-AMC. The reaction was stopped by the addition of 1 mM E-64 and incubation for 10 min at room temperature before measuring total fluorescence.…”

Subclassification and Biochemical Analysis of Plant Papain-Like Cysteine Proteases Displays Subfamily-Specific Characteristics

“…1A, marked with asterisks). These studies concern papain, chymopapain, and caricain from papaya (Carica papaya; Drenth et al, 1968;Katerelos et al, 1996;Maes et al, 1996), CysEP of castor bean (Ricinus communis; Than et al, 2004), actinidin of kiwi (Actinidia deliciosa; Varughese et al, 1992), ervatamin-A of crape jasmine (Ervatamia coronaria; Ghosh et al, 2008), and EP-B2 of barley (Hordeum vulgare; Bethune et al, 2006).…”

Structural basis of inhibition of cysteine proteases by E-64 and its derivatives

“…Table I lists some selected atomic distances between E-64 and the S n subsites of papain (a), actinidin (b), cathepsin L (c), and cathepsin K (d). The binding modes of E-64 to the respective S n subsites are schematically shown in Figure 2(a)-(d); caricain is a papain-family cysteine protease and its D158E mutant form complexed with E-64 has been analyzed by x-ray crystallography 10,11 ; however, data are not shown since the binding mode of this complex is analogous, so far as the inhibitor is visible in the caricain complex, to that in the papain and actinidin complexes. The C2 atom of E-64 epoxy ring is covalently bonded to the Cys-25 S ␥ atom in each active site; the C2-S ␥ bond distance is about 1.82 Å.…”