Analysis of the S(2) subsite specificities of the recombinant cysteine proteinases CPB of Leishmania mexicana, and cruzain of Trypanosoma cruzi, using fluorescent substrates containing non-natural basic amino acids.
We have explored the specificity of the S(2) subsite of recombinant cysteine proteinases from Leishmania mexicana (CPB2.8 Delta CTE) and from Trypanosoma cruzi (cruzain) employing a series of fluorogenic substrates based on the peptide Bz-F-R-MCA, in which Bz is the benzoyl group and the Phe residue has been substituted for by Arg, His and non-natural basic amino acids that combine a basic group with an aromatic or hydrophobic group at the side chain: 4-aminomethyl-phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). Bz-F-R-MCA was hydrolyzed well by CPB2.8 Delta CTE and cruzain, but all the substitutions of Phe resulted in less susceptible substrates for the two enzymes. CPB2.8 Delta CTE has a restricted specificity to hydrophobic side chains as with cathepsin L. However, the peptides with the residues Amf and Ama presented higher affinity to CPB2.8 Delta CTE, and the latter was an inhibitor of the enzyme. Although, cruzain accepts basic as well as hydrophobic residues at the S(2) subsite, it is more restrictive than cathepsin B and no inhibitor was found amongst the examined peptides.
Alves
L C
LC
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Tres de Maio 100, 04044-20 Sao Paulo, Brazil.
Melo
R L
RL
Cezari
M H
MH
Sanderson
S J
SJ
Mottram
J C
JC
Coombs
G H
GH
Juliano
L
L
Juliano
M A
MA
D024361:Amino Acids, Basic; D000818:Animals; D020134:Catalytic Domain; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D006868:Hydrolysis; D007894:Leishmania mexicana / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D024361:Amino Acids, Basic; D010455:Peptides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C074032:cruzain, Trypanosoma cruzi; D002401:Cathepsin B; D056668:Cathepsin L