Bioorganic Chemistry 30, 81–94 (2002) doi:10.1006/bioo.2001.1228, available online at http://www.idealibrary.com on Synthesis and Evaluation of Alternative Substrates for Arginase 1 Shoufa Han, Roger A. Moore, and Ronald E. Viola 2 Department of Chemistry, University of Toledo, Toledo, Ohio 43606 Received August 20, 2001 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 K m 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 k cat 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  -carboxyl group facilitates substrate binding, and the  -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.  2002 Elsevier Science (USA) Key Words: arginase; specificity; alternative substrates; thio analogs. INTRODUCTION Arginase is a ubiquitous enzyme that has been found in mammals (1), reptiles (2), plants (3), fungi (4), and bacteria (5). In mammals, arginase is found in liver (1) and also in nonheptatic tissues including red blood cells (6 ), lactating mammary gland, and kidney (1). Arginase is a trimeric manganese metalloenzyme that catalyzes the hydrolysis of L-arginine in the final step of the urea cycle, releasing urea and L-ornithine. In nonheptatic tissues, arginase plays a critical role in the biosynthesis of proline (7 ) and polyamines (8) by regulating the availability of L-ornithine. Arginase also regulates the biosynthesis of the cell-signaling molecule nitric oxide by affecting arginine availability, since both arginase and nitric oxide synthase share arginine as a common substrate (9). As a consequence of the reciprocal regulatory roles of arginase and nitric oxide synthase, arginase inhibition has therapeutic potential in treating nitric oxide-dependent smooth muscle disorders such as erectile dysfunction 1 This work was supported by a grant from the National Science Foundation (MCB0196103). 2 To whom correspondence and reprint requests should be addressed. Fax: 419-530-1583. E-mail: ron.viola@utoledo.edu. 81 0045-2068/02 $35.00  2002 Elsevier Science (USA) All rights reserved.