The substrate specificity of a recombinant cysteine protease from Leishmania mexicana: application of a combinatorial peptide library approach.
The substrate specificity of CPB2.8DeltaCTE, a recombinant cysteine protease from Leishmania mexicana, was mapped by screening a fluorescence-quenched combinatorial peptide library. Results from library screening indicated a preference for Arg or Lys in the S(3) subsite and for hydrophobic residues, both aliphatic and aromatic, in S(2). The S(1) subsite exhibited a specificity for the basic residues Arg and Lys. Generally, the specificity of the primed subsites was less strict compared with the non-primed side which showed preference for Arg, Lys and Ala in S'(1), Arg, Pro and Gly in S'(2) and Lys, Arg and Ser in S'(4). By contrast, a strict preference for the basic residues Arg and Lys was found for S'(3). Overall, there was a trend for basic residues in alternating subsites and smaller residues in the primed sites compared with the non-primed sites. In addition, there were strict requirements for the amino acids in subsites S(3)--S(1). Fluorescence-quenched peptides from the library with the highest on-resin cleavage were resynthesised and their kinetics of hydrolysis by CPB2.8DeltaCTE assessed in solution phase assays. Several good substrates containing the quintessential dipeptide particular to cathepsin-L-like enzymes, -F-R/K-, in P(2) and P(1) were identified (e.g. Y(NO(2))-EKFR down arrow RGK-K(Abz)G, Abz=2-aminobenzoyl; k(cat)K(m)(-1)=4298 mM(-1)s(-1)). However, novel substrates containing the dipeptide -L/I-Q- in P(2) and P(1) were also well hydrolysed (e.g. Y(NO(2))-YLQ down arrow GIQK-K(Abz)G; k(cat)K(m)(-1)=2583 mM(-1)s(-1)). The effect of utilising different fluorescent donor--quencher pairs on the value of k(cat)K(m)(-1) was examined. Generally, the use of the Abz/Q-EDDnp donor--quencher pair (EDDnp=N-(2,4-dinitrophenyl)ethylenediamine) instead of K(Abz)/Y(NO(2)) resulted in higher k(cat)K(m)(-1) values for analogous substrates.
St  Hilaire
P M
PM
Carlsberg Laboratory, Department of Chemistry Gamle Carlsberg Vej 10, 2500 Valby Denmark.
Alves
L C
LC
Sanderson
S J
SJ
Mottram
J C
JC
Juliano
M A
MA
Juliano
L
L
Coombs
G H
GH
Meldal
M
M
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D007894:Leishmania mexicana / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D015800:Protozoan Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D013379:Substrate Specificity
D000596:Amino Acids; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D019151:Peptide Library; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases