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Literature summary for 3.4.22.B49 extracted from

  • Stack, C.M.; Caffrey, C.R.; Donnelly, S.M.; Seshaadri, A.; Lowther, J.; Tort, J.F.; Collins, P.R.; Robinson, M.W.; Xu, W.; McKerrow, J.H.; Craik, C.S.; Geiger, S.R.; Marion, R.; Brinen, L.S.; Dalton, J.P.
    Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica (2008), J. Biol. Chem., 283, 9896-9908.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.4.22.B49

Cloned(Commentary)

Cloned (Comment) Organism
wild-type and variant enzymes are recombinantly expressed in Pichia pastoris Fasciola hepatica

Crystallization (Commentary)

Crystallization (Comment) Organism
the 1.4 A three-dimensional structure of the FheCL1 was determined by x-ray crystallography Fasciola hepatica

Protein Variants

Protein Variants Comment Organism
L205A mutation of FheCL1 markedly alters the activity profile from wild type enzyme. This variant exhibits a broader substrate specificity by accepting Phe, Trp, and Tyr at P2, residues that are not accepted by wild type FheCL1. kcat/Km for benzyloxycarbonyl-L-Phe-L-Arg-4-methylcoumarinyl-7-amide is 1.5fold higher than wild-type value, kcat/Km for benzyloxycarbonyl-L-Leu-L-Arg-4-methylcoumarinyl-7-amide is 2.5fold lower than wild-type value, kcat/Km for benzyloxycarbonyl-Pro-L-Arg-4-methylcoumarinyl-7-amide is 2.2fold lower than wild-type value, kcat/Km for tosyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide is 6.6fold lower than wild-type value, kcat/Km for tert-butoxycarbonyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide is 5fold than wild-type value Fasciola hepatica
L209A Ki value for benzyloxycarbonyl-Phe-Ala-diazomethyl ketone is 73fold lower than value for wild-type FheCL1 Fasciola hepatica
L67Y no significant change in the P2 preference to wild type FheCL1. This substitution does not alter the activity of the enzyme toward Pro in the P2 position. kcat/Km for benzyloxycarbonyl-L-Phe-L-Arg-4-methylcoumarinyl-7-amide is 2.3fold lower than wild-type value, kcat/Km for benzyloxycarbonyl-L-Leu-L-Arg-4-methylcoumarinyl-7-amide is 1.8fold lower than wild-type value, kcat/Km for benzyloxycarbonyl-Pro-L-Arg-4-methylcoumarinyl-7-amide is 1.2fold lower than wild-type value, kcat/Km for tosyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide is similar to wild-type value, kcat/Km for tert-butoxycarbonyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide is 1.8fold higher than wild-type value. Ki value for benzyloxycarbonyl-Phe-Ala-diazomethyl ketone is 12fold lower than wild-type value Fasciola hepatica

Inhibitors

Inhibitors Comment Organism Structure
benzyloxycarbonyl-Phe-Ala-diazomethyl ketone FheCL1 Fasciola hepatica
cathepsin K inhibitor II FheCL1 Fasciola hepatica

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.38
-
 benzyloxycarbonyl-L-Leu-L-Arg-4-methylcoumarinyl-7-amide mutant enzyme L67Y FheCL1 Fasciola hepatica
2.75
-
 benzyloxycarbonyl-L-Leu-L-Arg-4-methylcoumarinyl-7-amide mutant enzyme L205A FheCL1 Fasciola hepatica
4.35
-
 benzyloxycarbonyl-L-Leu-L-Arg-4-methylcoumarinyl-7-amide wild-type enzyme FheCL1 Fasciola hepatica
6.96
-
 tosyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide mutant enzyme L67Y FheCL1 Fasciola hepatica
8.16
-
 benzyloxycarbonyl-L-Phel-L-Arg-4-methylcoumarinyl-7-amide mutant enzyme L67Y FheCL1 Fasciola hepatica
10.02
-
 tosyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide wild-type enzyme FheCL1 Fasciola hepatica
10.43
-
 tert-butoxycarbonyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide wild-type enzyme FheCL1 Fasciola hepatica
11.13
-
 tert-butoxycarbonyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide mutant enzyme L67Y FheCL1 Fasciola hepatica
19.21
-
 benzyloxycarbonyl-L-Phel-L-Arg-4-methylcoumarinyl-7-amide mutant enzyme L205A FheCL1 Fasciola hepatica
20.35
-
 tosyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide mutant enzyme L205A FheCL1 Fasciola hepatica
21.57
-
 tert-butoxycarbonyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide mutant enzyme L205A FheCL1 Fasciola hepatica
24.18
-
 benzyloxycarbonyl-L-Phel-L-Arg-4-methylcoumarinyl-7-amide wild-type enzyme FheCL1 Fasciola hepatica
48.41
-
 benzyloxycarbonyl-L-Pro-L-Arg-4-methylcoumarinyl-7-amide mutant enzyme L205A FheCL1 Fasciola hepatica
137
-
 benzyloxycarbonyl-L-Pro-L-Arg-4-methylcoumarinyl-7-amide mutant enzyme L67Y FheCL1 Fasciola hepatica
191.2
-
 benzyloxycarbonyl-L-Pro-L-Arg-4-methylcoumarinyl-7-amide wild-type enzyme FheCL1 Fasciola hepatica

Organism

Organism UniProt Comment Textmining
Fasciola hepatica Q24940
-
-

Posttranslational Modification

Posttranslational Modification Comment Organism
proteolytic modification FhCL1 is expressed as 37000 Da zymogen that autocatalytically processes at pH 4.5 to produce a 24500 Da mature enzyme Fasciola hepatica

Purification (Commentary)

Purification (Comment) Organism
recombinant zymogen of cathepsin L1 Fasciola hepatica

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
benzyloxycarbonyl-L-Leu-L-Arg-4-methylcoumarinyl-7-amide + H2O FheCL1 Fasciola hepatica benzyloxycarbonyl-L-Leu-L-Arg + 7-amino-4-methylcoumarin
-
 ?
benzyloxycarbonyl-L-Phe-L-Arg-4-methylcoumarinyl-7-amide + H2O FheCL1 Fasciola hepatica benzyloxycarbonyl-L-Phe-L-Arg + 7-amino-4-methylcoumarin
-
 ?
benzyloxycarbonyl-L-Pro-L-Arg-4-methylcoumarinyl-7-amide + H2O FheCL1 Fasciola hepatica benzyloxycarbonyl-L-Pro-L-Arg + 7-amino-4-methylcoumarin
-
 ?
Collagen + H2O whereas FheCL1 produces clear degradation fragments, FheCL2 degrades the collagen completely, particularly at pH 4.0, indicating that only the latter cleaves efficiently within the helical structures Fasciola hepatica ?
-
 ?
additional information wild-type FheCL1 shows clear preference for Arg at P1. Other residues accommodated in this position including Lys, Glu, Thr, and Met are all cleaved at similar relative rates to that observed for human cathepsin L and cathepsin K. Similar results are obtained for the variants FheCL1 L67Y and FheCL1 L205A. FheCL1 shows distinct preference for hydrophobic amino acids in the P2, Leu is favored. FheCL1 and FheCL2 are similar to cathepsin K with regard to their preference for a P2 Leu over Phe (human cathepsin L has a preference for P2 Phe over Leu). Both enzymes can accommodate Pro in the P2 position, but this is more readily accepted by FheCL2 compared with FheCL1. Neither enzyme, however, cleaves substrates with this residue in the P2 position as readily as human cathepsin K Fasciola hepatica ?
-
 ?
tert-butoxycarbonyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide + H2O FheCL1 Fasciola hepatica tert-butoxycarbonyl-Gly-Pro-Arg + 7-amino-4-methylcoumarin
-
 ?
tosyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide + H2O FheCL1 Fasciola hepatica tosyl-Gly-Pro-Arg + 7-amino-4-methylcoumarin
-
 ?

Synonyms

Synonyms Comment Organism
cathepsin L1
-
 Fasciola hepatica
FheCL1
-
 Fasciola hepatica

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.113
-
 tosyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide mutant enzyme L205A FheCL1 Fasciola hepatica
0.122
-
 benzyloxycarbonyl-L-Pro-L-Arg-4-methylcoumarinyl-7-amide mutant enzyme L205A FheCL1 Fasciola hepatica
0.2
-
 tert-butoxycarbonyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide mutant enzyme L205A FheCL1 Fasciola hepatica
0.26
-
 tosyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide mutant enzyme L67Y FheCL1 Fasciola hepatica
0.36
-
 tosyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide wild-type enzyme FheCL1 Fasciola hepatica
0.48
-
 tert-butoxycarbonyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide wild-type enzyme FheCL1 Fasciola hepatica
0.62
-
 benzyloxycarbonyl-L-Pro-L-Arg-4-methylcoumarinyl-7-amide mutant enzyme L67Y FheCL1 Fasciola hepatica
0.93
-
 tert-butoxycarbonyl-Gly-Pro-Arg-4-methylcoumarinyl-7-amide mutant enzyme L67Y FheCL1 Fasciola hepatica
1.03
-
 benzyloxycarbonyl-L-Pro-L-Arg-4-methylcoumarinyl-7-amide wild-type enzyme FheCL1 Fasciola hepatica
1.73
-
 benzyloxycarbonyl-L-Leu-L-Arg-4-methylcoumarinyl-7-amide mutant enzyme L67Y FheCL1 Fasciola hepatica
3.58
-
 benzyloxycarbonyl-L-Phe-L-Arg-4-methylcoumarinyl-7-amide mutant enzyme L67YFheCL1 Fasciola hepatica
9.15
-
 benzyloxycarbonyl-L-Leu-L-Arg-4-methylcoumarinyl-7-amide mutant enzyme L205A FheCL1 Fasciola hepatica
24.69
-
 benzyloxycarbonyl-L-Phe-L-Arg-4-methylcoumarinyl-7-amide wild-type enzyme, FheCL1 Fasciola hepatica
29.6
-
 benzyloxycarbonyl-L-Phe-L-Arg-4-methylcoumarinyl-7-amide mutant enzyme L205A FheCL1 Fasciola hepatica
36.52
-
 benzyloxycarbonyl-L-Leu-L-Arg-4-methylcoumarinyl-7-amide wild-type enzyme FheCL1 Fasciola hepatica

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.0000108
-
 cathepsin K inhibitor II wild-type enzyme, FheCL1 Fasciola hepatica
0.0000133
-
 cathepsin K inhibitor II mutant enzyme L67Y, FheCL1 Fasciola hepatica
0.0000154
-
 benzyloxycarbonyl-Phe-Ala-diazomethyl ketone mutant enzyme L209A, FheCL1 Fasciola hepatica
0.0000936
-
 benzyloxycarbonyl-Phe-Ala-diazomethyl ketone mutant enzyme L67Y, FheCL1 Fasciola hepatica
0.000116
-
 cathepsin K inhibitor II mutant enzyme L209A, FheCL1 Fasciola hepatica
0.001125
-
 benzyloxycarbonyl-Phe-Ala-diazomethyl ketone wild-type enzyme, FheCL1 Fasciola hepatica