Fig 1.
The structure of papain and its precursor form.
a) A schematic representation of papain precursor and activation mechanism. b) Three dimentional structure of zymogen of papain (PDB ID: 3TNX); catalytic domain is represented as electrostatic potential surface with catalytic dyad residues represented as light-blue CPK model. The pro-peptide is represnted in ribbon. The color scheme used for (a) and (b) are: nevyblue, signal peptide; green, N-terminal pro-domain; yellow, PBL binding β-strand; magenta, the polypeptide blocking the substrate binding subsites; orange, autocatalytic cleavage site; blue-red gradient; catalytic domain. ‘m’ and ‘p’ tags in the sequence number represent mature and pro-peptide region respectively.
Table 1.
Oligonucleotides used for site-directed mutagenesis.
Table 2.
Kinetic constants for peptidyl p-nitroanilides.
Each kinetic constant with standard deviation is the average value of three independent experiments. Highest kcat/ Km (M-1 sec-1) value for each substrate is shown in bold.
Table 3.
Data collection and refinement statistics.
Fig 2.
SDS-PAGE analyses of the proteases.
a) Purified pro-proteases. b) Gelatin gel zymography for WT (TS) and three mutants. 2μg pro-proteases were loaded in each lane. The lanes of each gel are marked at the bottom. M lane is for molecular weight marker whose values in kDa are given at the left panel.
Fig 3.
a) Reducing SDS-PAGE analysis of the generated mature proteases. b) Far-UV CD spectra of mature proteases in the range 190 to 250 nm. c) MALDI-TOF MS spectra of mature proteases as shown in the SDS-PAGE (panel a). d) Peptide mass finger printing analyses after trypsin in-gel digestion of I86L and WT (TS) mature protease. e) Theoretical mass and sequence of the N-terminus of expected cleavages upto 7 residue downstream of the N-terminus of mature papain from plant latex. Sequences marked in red and green correspond to the peaks marked by same colored arrows in panel d. Red and green arrows represent high and low intensity peaks respectively in panel d. f) Sequence of the recombinant WT (TS); yellow, blue and black colour represent vector tag, pro-peptide and mature domain sequence respectively. Red and green arrows indicate cleavages corresponding to the peptide masses indicated in the same color in MS experiment (panels d and e). The sequences which matched with mature domain obtained from MS/MS analyses are underlined.
Fig 4.
Effect of substitution at residue 86 of pro-peptide of papain on its proteolytic acivity.
a) relative [normalised to WT (TS)] specific activity values against azo-casein and relative Kcat/Km values for the substrate Pyroglutamyl-Phe-Leu-p-nitroanilide. b) The actual values of the same are given in the table. c) Michaelis-Menten plot of proteolytic activity against different substrates for three mutants and WT (TS) papain. The error bar of each points represents standard error of the mean of replicates.
Fig 5.
X-ray structures of the mutants and WT (TS).
(a) Omit map (Fo-Fc) of I86L and I86F. The omit maps, contoured at 3.5σ level, were calculated by omitting the pro-peptide blocking the catalytic cleft. Catalytic domains of the two mutants are represented n electrostatic surfaces with the catalytic dyad in orange sphere superimposed therein. (b) Superposition of pro-peptide of I86L, I86F mutants and WT (TS). The propeptides are represented in ribbon. I86L, coloured in green; I86F, coloured in light blue; WT (TS) in light orange. The mature domain of the WT (TS) is in surface presentation with catalytic dyad in ball and stick. (c) Superposition of the mature catalytic domain with same color code used in (b). (d) and (e) The 2mFo-DFc electron density map at 1.5σ level of residues Tyr168, Tyr174 and Val220 in three proteins. The co-ordinates and structure factors of the WT (TS) have been taken from pdb_id 3TNX. ‘m’ and ‘p’ tags in the sequence number represent mature and pro-peptide region respectively.
Fig 6.
Structural comparison of catalytic cleft structures of I86F and I86L with that of the WT (TS).
The catalytic domains are in surface presentation; I86L, coloured in green; I86F, coloured in light blue; WT (TS) in light orange. The pro-peptide parts blocking the catalytic clefts are presented as stick model with Carbon atoms coloured as their respective catalytic domain surface colour. a) and b) Superposition of I86L and I86F mutants with the WT (TS) respectively. c) Individual I86L, I86F and WT (TS) in a similar orientation of a) and b). The arrow indicates a cavity formed by two residues Tyr168 and Tyr174. d) Superposition of I86L, I86F and WT (TS) showing some residues of the catalytic cleft responsible for micro-changes in the cleft conformation of the mutants. The catalytic dyad residues are C132A and H266. The Cɑ trace is drawn on WT (TS)structure with mature domain in light orange and pro-peptide in magenta. The position of pro-peptide also represents the flow of substrate during Michaelis comlex formation.The co-ordinates and structure factors of the WT (TS) have been taken from pdb_id 3TNX. ‘m’ and ‘p’ tags in the sequence number represent mature and pro-peptide region respectively.