We investigated whether an uncharacterized protein from guinea pig could be the enzyme behind Kidd's serendipitous discovery, made over 60 years ago, that guinea pig serum has cell killing ability. It has been long known that an enzyme with l-asparaginase activity is responsible for cell killing, although astonishingly, its identity remains unclear. Bacterial asparaginases with similar cell killing properties have since become a mainstay therapy of certain cancers such as acute lymphoblastic leukemia. By hydrolyzing asparagine to aspartate and ammonia, these drugs deplete the asparagine present in the blood, killing cancer cells that rely on extracellular asparagine uptake for survival. However, bacterial ... More
We investigated whether an uncharacterized protein from guinea pig could be the enzyme behind Kidd's serendipitous discovery, made over 60 years ago, that guinea pig serum has cell killing ability. It has been long known that an enzyme with l-asparaginase activity is responsible for cell killing, although astonishingly, its identity remains unclear. Bacterial asparaginases with similar cell killing properties have since become a mainstay therapy of certain cancers such as acute lymphoblastic leukemia. By hydrolyzing asparagine to aspartate and ammonia, these drugs deplete the asparagine present in the blood, killing cancer cells that rely on extracellular asparagine uptake for survival. However, bacterial asparaginases can elicit an adverse immune response. We propose that replacement of bacterial enzymes with the guinea pig asparaginase responsible for serum activity, by its virtue of being more closely related to human enzymes, will be less immunogenic. To this goal, we investigated whether an uncharacterized protein from guinea pig with putative asparaginase activity, which we call gpASNase3, could be that enzyme. We examined its self-activation process (gpASNase3 requires autocleavage to become active), kinetically characterized it for asparaginase and β-aspartyl dipeptidase activity, and elucidated its crystal structure in both the uncleaved and cleaved states. This work reveals that gpASNase3 is not the enzyme responsible for the antitumor effects of guinea pig serum. It exhibits a low affinity for asparagine as measured by a high Michaelis constant, KM, in the millimolar range, in contrast to the low KM (micromolar range) required for asparaginase to be effective as an anticancer agent.
