Human 60-kDa lysophospholipase contains an N-terminal L-asparaginase domain that is allosterically regulated by L-asparagine.

The structural and functional characterization of human enzymes that are of potential medical and therapeutic interest is of prime significance for translational research. One of the most notable examples of a therapeutic enzyme is L-asparaginase, which has been established as an antileukemic protein drug for more than four decades. Up until now, only bacterial enzymes have been used in therapy despite a plethora of undesired side effects mainly attributed to the bacterial origins of these enzymes. Therefore, the replacement of the currently approved bacterial drugs by human homologs aiming at the elimination of adverse effects is of great importance. Recently, we structurally and biochemically characterized the enzyme human L-asparaginase 3 (hASNase3), which possesses L-asparaginase activity and belongs to the N-terminal nucleophile superfamily of enzymes. Inspired by the necessity for the development of a protein drug of human origin, in the present study, we focused on the characterization of another human L-asparaginase, termed hASNase1. This bacterial-type cytoplasmic L-asparaginase resides in the N-terminal subdomain of an overall 573-residue protein previously reported to function as a lysophospholipase. Our kinetic, mutagenesis, structural modeling, and fluorescence labeling data highlight allosteric features of hASNase1 that are similar to those of its Escherichia coli homolog, EcASNase1. Differential scanning fluorometry and urea denaturation experiments demonstrate the impact of particular mutations on the structural and functional integrity of the L-asparaginase domain and provide a direct comparison of sites critical for the conformational stability of the human and E. coli enzymes.