This year, over 120,000 people are predicted to be diagnosed with either melanoma, renal, or hepatocellular carcinoma, among the most malicious forms of cancer. The treatments available are limited and only mildly effective. However, one common characteristic of these cancer cells exposes a promising avenue for treatment: they are arginine auxotrophs, stemming from their deficiency in arginosuccinate synthetase. These cancers are unable to synthesize this important amino acid building block; thus, they must import arginine from the extracellular environment. Already, extensive research has established that sustained systemic depletion of extracellular arginine constitutes a powerful strategy for selective killing of these cancers. A PEGylated bacterial enzyme, Arginine Deiminase, is able to accomplish long-term systemic arginine deprivation, but immunogenic responses are a major drawback with exogenous protein therapeutics.

An alternative candidate is human Arginase I, a manganese metalloenzyme localized to the liver which plays an integral role in the urea cycle, catalyzing the hydrolysis of L-arginine into L-ornithine and urea. Although it lacks immogenicity, it fails to satisfy other criteria for an effective therapeutic, characterized by low substrate affinity, and non-optimal activity under physiological conditions,

We sought to address these drawbacks and ultimately enhance the therapeutic value of Arginase I. Simple replacement with cobalt led to an unexpected 10 fold increase in catalytic activity at physiological pH. Kinetic analysis revealed this to originate from cobalt's ability to depress the pKa of the active site hydroxide by >2 units, permitting more of the reactive nucleophile to be present at neutral pH. Cobalt substitution also increases the affinity for substrate and product ligands. Building upon recent work that described enhanced arginase activity and stability when residue Cys303 is S-nitrosylated, we performed saturation mutagenesis at this position followed by screening for activity. Characterization of variants revealed that a C303P substitution conferred a 60 % improvement in serum stability. Additionally, we explored fine-tuning the active site through directed mutagenesis of selected first and second shell metal ligands. This led to the isolation of S230G and S230C second-shell substitutions, variants that display a 20 fold improvement in kcat /Km over native Mn-arginase. Finally, we tested Mn-arginase, Co-arginase, and Co-C303P-arginase for their ability to kill Hep3b hepatocarcinoma cells and found that Co-arginase was 10-fold more potent that Mn-arginase while Co-C303P-arginase was about 30-fold better. Armed with these kinetically optimized variants, efforts will now be directed towards improving stability in vivo

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