Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II.
Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer-dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia.
Institute of Physiological Chemistry, Philipps University, Marburg, Germany.
Shikha
S
Verma
School of Biotechnology, KIIT University, Bhubaneswar 751024, Orissa, India.
Ranjit Kumar
RK
Mehta
Imgenex India Pvt. Ltd, Bhubaneswar 751024, Orissa, India.
Prasanta
P
Maiti
Institute of Physiological Chemistry, Philipps University, Marburg, Germany.
Klaus-Heinrich
KH
Röhm
School of Biotechnology, KIIT University, Bhubaneswar 751024, Orissa, India. Electronic address: asonawane@kiitbiotech.ac.in.
Avinash
A
Sonawane
D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011487:Protein Conformation; D011489:Protein Denaturation
D001215:Asparaginase
Activity; Asparaginase; E. coli; Mutagenesis; Stability