Synthesis and Evaluation of Alternative Substrates for Arginasease

Two novel carboxyl-containing arginase substrates, 4-guanidino-3-nitrobenzoic acid and 4-guanidino-2-nitrophenylacetic acid, have been synthesized and found to give enhanced catalysis and dramatically lower Km values relative to 1-nitro-3-guanidinobenzene, a substrate designed for use in a chromophoric arginase assay. To more efficiently mimic the natural substrate, a series of sulfur analogs of l-arginine were synthesized and kinetically characterized. The parent compound, l-thioarginine, with the bridging guanidinium nitrogen of l-arginine replaced with sulfur, functions as efficiently as the natural substrate. The desamino analog shows extremely low turnover, while the kcat of the descarboxy analog is only 75-fold lower than that of arginine. These results suggest that the bridging nitrogen of l-arginine is not important for either substrate binding or catalysis, while the α-carboxyl group facilitates substrate binding, and the α-amino group is necessary for efficient catalysis. Isothiourea homologs previously reported to be nitric oxide synthase inhibitors have been found to undergo a rapid non-enzymatic rearrangement to a species that is probably the true inhibitor.