Activating Compound | Comment | Organism | Structure |
---|---|---|---|
additional information | activation of ananain is carried out in the activity assay buffer containing 20 mM sodium acetate, pH 5.0, 300 mM KCl, 1 mM EDTA, 10 mM L-cysteine at 37°C for 15 min | Ananas comosus |
Crystallization (Comment) | Organism |
---|---|
purified native enzyme free and in complex with inhibitor E-64, hanging drop vapor diffusion technique, mixing of 10 mg/ml protein in 20 mM Tris, pH 8.0, and 20 mM NaCl with reservoir solution containing 72% w/v MPD, and 0.1 M Tris, pH 8.8, 20°C, 2 weeks, the enzyme-inhibitor complex is built from activated ananain mixed with inhibition buffer containing 20 mM sodium acetate, pH 5.5, 300 mM KCl, 1 mM EDTA, 10 mM L-cysteine, 5% v/v DMSO, with 0.5 mM E-64 at a final concentration of 0.1 mM and incubation at room temperature for 3 h. The inhibited ananain is desalted and equilibrated with 20 mM Tris, pH 8.0, and 20 mM NaCl and then concentrated to 6.5 mg/ml, crystals of E-64-ananain are obtained in the same condition as for unbound ananain, X-ray diffraction structure determination and analysis at 1.73 and 1.98 A resolution, respectively | Ananas comosus |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
E-64 | high inhibitory efficiency leading to complete inhibition, binding structure and interaction analysis, molecular mechanism, overview. Of the trans-epoxysuccinic acid moiety of E-64, the C2 atom is covalently bound to the active site Cys25 Sgamma, irreversibly inhibiting the catalytic activity of Cys25. Accommodation of E-64 in the substrate binding groove of ananain does not cause any major structural alteration of ananain | Ananas comosus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetic analysis | Ananas comosus | |
0.00394 | - |
PLR | pH 7.4, 37°C | Ananas comosus | |
0.00415 | - |
ALR | pH 7.4, 37°C | Ananas comosus | |
0.00429 | - |
VLR | pH 7.4, 37°C | Ananas comosus | |
0.0045 | - |
PLN | pH 7.4, 37°C | Ananas comosus | |
0.00536 | - |
VLK | pH 7.4, 37°C | Ananas comosus | |
0.00585 | - |
ALK | pH 7.4, 37°C | Ananas comosus | |
0.00592 | - |
PLQ | pH 7.4, 37°C | Ananas comosus | |
0.00694 | - |
PLK | pH 7.4, 37°C | Ananas comosus | |
0.0105 | - |
benzyloxycarbonyl-Phe-Val-Arg-7-amido-4-methylcoumarin | pH 7.4, 37°C | Ananas comosus | |
0.018 | - |
benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin | pH 7.4, 37°C | Ananas comosus | |
0.198 | - |
benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin | pH 7.4, 37°C | Ananas comosus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Ananas comosus | - |
- |
- |
Purification (Comment) | Organism |
---|---|
native enzyme from stem crude bromelain by cation exchange chromatography, gel filtration, and metal affinity chromatography | Ananas comosus |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
commercial preparation | active purified ananain | Ananas comosus | - |
stem | - |
Ananas comosus | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ALK + H2O | - |
Ananas comosus | ? | - |
? | |
ALR + H2O | - |
Ananas comosus | ? | - |
? | |
benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin + H2O | - |
Ananas comosus | benzyloxycarbonyl-Arg-Arg + 7-amino-4-methylcoumarin | - |
? | |
benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin + H2O | - |
Ananas comosus | benzyloxycarbonyl-Phe-Arg + 7-amino-4-methylcoumarin | - |
? | |
benzyloxycarbonyl-Phe-Val-Arg-7-amido-4-methylcoumarin + H2O | - |
Ananas comosus | benzyloxycarbonyl-Phe-Val-Arg + 7-amino-4-methylcoumarin | - |
? | |
additional information | peptidyl substrate specificity analysis of ananain, detailed overview. The optimal tripeptide is PLQ with cleavage occurring after the Gln residue. Fluorescent enzyme assay method. The PLQ and VLR substrates are found to be the optimal substrates for cleavage by ananain, with a similar kcat/KM value. Substituting the P1-Arg residue of PLR, VLR, and ALR with a Lys only slightly lowers their kcat/KM values, whilst replacing the P1-Gln residue of PLQ with an Asn causes a 20fold decrease in the kcat/KM value | Ananas comosus | ? | - |
? | |
PLK + H2O | - |
Ananas comosus | ? | - |
? | |
PLN + H2O | - |
Ananas comosus | ? | - |
? | |
PLQ + H2O | - |
Ananas comosus | ? | - |
? | |
PLR + H2O | - |
Ananas comosus | ? | - |
? | |
VLK + H2O | - |
Ananas comosus | ? | - |
? | |
VLR + H2O | - |
Ananas comosus | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 25000, about | Ananas comosus |
More | ananain exhibits a typical papain-like domain organization: an alpha-helix abundant L-domain comprised of residues 10-111 and 208-215, and a beta-sheet rich R-domain consisting of residues 1-9 and 112-207. In between the two domains there are two pocket-like structures, marked as pocket 1 and pocket 2. Pocket 1 is formed by the side chains of Gln19, Gly23, Trp26, Gly64, Trp180 and most importantly, the active site residues Cys25 and His157, the latter two residues essentially forming a catalytic diad. In contrast to pocket 1, which is geometrically flat and open, pocket 2 is deep and narrow, formed by the side chains of a number of hydrophobic residues, including Trp26, Trp66, Ile67, Ala132, Leu155 and Ala158 | Ananas comosus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Ananas comosus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.16 | - |
benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin | pH 7.4, 37°C | Ananas comosus | |
0.36 | - |
PLN | pH 7.4, 37°C | Ananas comosus | |
1.58 | - |
benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin | pH 7.4, 37°C | Ananas comosus | |
3.58 | - |
ALK | pH 7.4, 37°C | Ananas comosus | |
4.96 | - |
ALR | pH 7.4, 37°C | Ananas comosus | |
5.62 | - |
PLR | pH 7.4, 37°C | Ananas comosus | |
6.08 | - |
PLK | pH 7.4, 37°C | Ananas comosus | |
6.29 | - |
VLK | pH 7.4, 37°C | Ananas comosus | |
7.13 | - |
VLR | pH 7.4, 37°C | Ananas comosus | |
9.8 | - |
PLQ | pH 7.4, 37°C | Ananas comosus | |
13.9 | - |
benzyloxycarbonyl-Phe-Val-Arg-7-amido-4-methylcoumarin | pH 7.4, 37°C | Ananas comosus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
5 | 7.4 | assay at | Ananas comosus |
General Information | Comment | Organism |
---|---|---|
evolution | despite a high degree of sequence identity between ananain and stem bromelain, the most abundant bromelain cysteine protease, ananain displays distinct chemical properties, substrate preference and inhibitory profile compared to stem bromelain. Ananain belongs to the papain family of peptidases with a high degree of identity to chymopapain (52%) and papain (60%) from papaya | Ananas comosus |
additional information | the enzyme shows a geometrically flat and open S1 subsite for ananain. This subsite accommodates diverse P1 substrate residues, while a narrow and deep hydrophobic pocket-like S2 subsite would accommodate a non-polar P2 residue, such as the preferred Leu residue observed in specificity studies | Ananas comosus |
physiological function | ananain accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain | Ananas comosus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.8 | - |
benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin | pH 7.4, 37°C | Ananas comosus | |
6 | 12 | ALK | pH 7.4, 37°C | Ananas comosus | |
80 | - |
PLN | pH 7.4, 37°C | Ananas comosus | |
87.8 | - |
benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin | pH 7.4, 37°C | Ananas comosus | |
876.1 | - |
PLK | pH 7.4, 37°C | Ananas comosus | |
1173.5 | - |
VLK | pH 7.4, 37°C | Ananas comosus | |
1195.2 | - |
ALR | pH 7.4, 37°C | Ananas comosus | |
1323.8 | - |
benzyloxycarbonyl-Phe-Val-Arg-7-amido-4-methylcoumarin | pH 7.4, 37°C | Ananas comosus | |
1426.4 | - |
PLR | pH 7.4, 37°C | Ananas comosus | |
1655.4 | - |
PLQ | pH 7.4, 37°C | Ananas comosus | |
1662 | - |
VLR | pH 7.4, 37°C | Ananas comosus |