The Thiol Proteinases from the Latex of<i>Carica papaya</i>L. II. The Primary Structure of Proteinase Ω

Dubois, Thierry; Kleinschmidt, Traute; Schnek, Arthur G.; Looze, Yvan; Braunitzer, Gerhard

doi:10.1515/bchm3.1988.369.2.741

Cited by 65 publications

(27 citation statements)

References 16 publications

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“…Caricain (previously known as papaya solubilized in ethanol and any non-bound E-64 was removed by washprotease omega) is the most basic cysteine protease from the ing over a dialysis membrane. latex of Carica papaya with 69.5% sequence identity with papain [6]. Refined structures of the papain superfamily are:…”

Section: Production Of the Caricain Mutant Di58e/e-64 Complex Was Solmentioning

confidence: 99%

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

et al. 1996

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'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.

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Section: Production Of the Caricain Mutant Di58e/e-64 Complex Was Solmentioning

confidence: 99%

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

et al. 1996

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“…[4][5][6] Recent evidence, however, supports the idea that MG may also possess well-defined tertiary contacts. [7][8][9] The stem bromelain (SB), from Ananas cosmosus, like other cysteine proteinases, belongs to the þ protein class, and the highly similar amino acid sequences of papain, 10 actinidin, 11 proteinase O, 12 chymopapain, 13 and SB 14 indicate that the polypeptide chains of these proteins may share a common folding pattern. This has been substantiated for the former three proteinases by detailed X-ray diffraction studies.…”

Section: Introductionmentioning

confidence: 99%

Low versus high molecular weight poly(ethylene glycol)‐induced states of stem bromelain at low pH: Stabilization of molten globule and unfolded states

et al. 2005

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The effect of low, medium, and high molecular weight poly(ethylene glycol) (e.g., PEG-400, -6000, and -20,000) on the structure of the acid unfolded state of unmodified stem bromelain (SB) obtained at pH 2.0 has been studied by various spectroscopic methods. The conformation of stem bromelain at pH 2.0 exhibits substantial loss of secondary structure and almost complete loss of native tertiary contacts and has been termed the acid unfolded state (A(U)). Addition of PEG-400 to A(U) led to an increase in the mean residue ellipticity (MRE) value at 222 nm, indicating formation of alpha-helical structure. On the other hand, PEG-6000 and 20,000 led to a decrease in the MRE value at 222 nm, indicating unfolding of the A(U) state. Interestingly, at 70% (w/v) PEG-400 and 40% (w/v) PEG-20,000, MRE values at 222 nm almost approach the native state at pH 7.0 and the unfolded state (6 M GnHCl) of stem bromelain, respectively. The probes for tertiary structure showed formation of nonnative tertiary contacts in the presence of 70% (w/v) PEG-400, while 40% (w/v) PEG-6000 and 20,000 were found to stabilize the unfolded state of SB. An increase in binding of 1-anilino 8-naphthalene sulfonic acid and a decrease in fractional accessibility of tryptophan residues (f(a)) compared to A(U) in the presence of 70% PEG-400 indicate that the PEG-400-induced state has a significant amount of exposed hydrophobic patches and is more compact than A(U). The results imply that the PEG-400-induced state has characteristics of molten globule, and higher molecular weight PEGs led to the unfolding of the A(U) state.

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“…This finding supports the notion that plant extracellular proteinases may be part of a Latex of Carica papaya L. contains a mixture of cys-signaling process involving the intracellular MAP kiteine endopeptidases (1), such as papain (EC 3.4.22.2) nase pathway. We are investigating the identity of each (2), chymopapains A and B (3.4.22.6) (3), papaya endo-of these proteolytic activities in latex from C candamarpeptidase III, papaya endopeptidase IV (4,5), and encensis to learn about their role during clot formation dopeptidase ⍀ (caricain) (6). Fresh or dried latex oband analyzing their similarities with known compotained by injuring leaves or fruits of C. papaya contains nents of the transduction signal pathway.…”

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confidence: 99%