Literature summary for 3.4.22.31 extracted from

Yongqing, T.; Wilmann, P.G.; Pan, J.; West, M.L.; Brown, T.J.; Mynott, T.; Pike, R.N.; Wijeyewickrema, L.C.
Determination of the crystal structure and substrate specificity of ananain (2019), Biochimie, 166, 194-202 .

Search for more literature for 3.4.22.31

Activating Compound

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 (Commentary)

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

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

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 [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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

Organism	UniProt	Comment	Textmining
Ananas comosus	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
native enzyme from stem crude bromelain by cation exchange chromatography, gel filtration, and metal affinity chromatography	Ananas comosus

Source Tissue

Source Tissue	Comment	Organism	Textmining
commercial preparation	active purified ananain	Ananas comosus	-
stem	-	Ananas comosus	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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 [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Ananas comosus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
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

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
5	7.4	assay at	Ananas comosus

General Information

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 [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
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