Critical role of amino acid 23 in mediating activity and specificity of vinckepain-2, a papain-family cysteine protease of rodent malaria parasites.
Cysteine proteases of Plasmodium falciparum, known as falcipains, have been identified as haemoglobinases and potential drug targets. As anti-malarial drug discovery requires the analysis of non-primate malaria, genes encoding related cysteine proteases of the rodent malaria parasites P. vinckei (vinckepain-2) and P. berghei (berghepain-2) were characterized. These genes encoded fairly typical papain-family proteases, but they contained an unusual substitution of Gly23 with Ala (papain numbering system). Vinckepain-2 was expressed in Escherichia coli, solubilized, refolded and autoprocessed to an active enzyme. The protease shared important features with the falcipains, including an acidic pH optimum, preference for reducing conditions, optimal cleavage of peptide substrates with P2 Leu and ready hydrolysis of haemoglobin. However, key differences between the plasmodial proteases were identified. In particular, vinckepain-2 showed very different kinetics against many substrates and an unusual preference for peptide substrates with P1 Gly. Replacement of Ala23 with Gly remarkably altered vinckepain-2, including loss of the P1 Gly substrate preference, markedly increased catalytic activity ( k cat/ K m increased approx. 100-fold) and more rapid autohydrolysis. The present study identifies key animal-model parasite targets. It indicates that drug discovery studies must take into account important differences between plasmodial proteases and sheds light on the critical role of amino acid 23 in catalysis by papain-family proteases.
Singh
Ajay
A
Department of Medicine, San Francisco General Hospital, San Francisco, CA 94143, USA.
Shenai
Bhaskar R
BR
Choe
Youngchool
Y
Gut
Jiri
J
Sijwali
Puran S
PS
Craik
Charles S
CS
Rosenthal
Philip J
PJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015801:Helminth Proteins; D008288:Malaria / Q000469:parasitology; D008969:Molecular Sequence Data; D009154:Mutation; D010206:Papain / Q000737:chemistry; D019151:Peptide Library; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D017510:Protein Folding; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012376:Rodent Diseases / Q000469:parasitology; D012377:Rodentia; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D015801:Helminth Proteins; D019151:Peptide Library; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C469677:vinckepain 2, Plasmodium falciparum; D010206:Papain