Recombinant L-asparaginase 1 from Saccharomyces cerevisiae: an allosteric enzyme with antineoplastic activity.
L-asparaginase (L-ASNase) (EC 3.5.1.1) is an important enzyme for the treatment of acute lymphoblastic leukaemia. Currently, the enzyme is obtained from bacteria, Escherichia coli and Erwinia chrysanthemi. The bacterial enzymes family is subdivided in type I and type II; nevertheless, only type II have been employed in therapeutic proceedings. However, bacterial enzymes are susceptible to induce immune responses, leading to a high incidence of adverse effects compromising the effectiveness of the treatment. Therefore, alternative sources of L-ASNase may be useful to reduce toxicity and enhance efficacy. The yeast Saccharomyces cerevisiae has the ASP1 gene responsible for encoding L-asparaginase 1 (ScASNase1), an enzyme predicted as type II, like bacterial therapeutic isoforms, but it has been poorly studied. Here we characterised ScASNase1 using a recombinant enzyme purified by affinity chromatography. ScASNase1 has specific activity of 196.2 U/mg and allosteric behaviour, like type I enzymes, but with a low K0.5= 75 μM like therapeutic type II. We showed through site-directed mutagenesis that the T64-Y78-T141-K215 residues are involved in catalysis. Furthermore, ScASNase1 showed cytotoxicity for the MOLT-4 leukemic cell lineage. Our data show that ScASNase1 has characteristics described for the two subfamilies of l-asparaginase, types I and II, and may have promising antineoplastic properties.
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo/SP 05508-000, Brazil.
Iris Munhoz
IM
Costa
Biosciences Institute, São Paulo State University - UNESP, Coastal Campus, São Vicente/SP 11330-900, Brazil.
Leonardo
L
Schultz
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo/SP 05508-000, Brazil.
Beatriz
B
de Araujo Bianchi Pedra
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo/SP 05508-000, Brazil.
Mariana Silva Moreira
MS
Leite
Department of Clinical and Toxicological Analysis School of Pharmaceutical Sciences, University of São Paulo, São Paulo/SP 05508-000, Brazil.
Sandra H P
SH
Farsky
Biosciences Institute, São Paulo State University - UNESP, Coastal Campus, São Vicente/SP 11330-900, Brazil.
Marcos Antonio
MA
de Oliveira
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo/SP 05508-000, Brazil.
Adalberto
A
Pessoa
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo/SP 05508-000, Brazil.
Gisele
G
Monteiro
D000494:Allosteric Regulation; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001216:Asparagine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D000970:Antineoplastic Agents; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D001216:Asparagine