Synthesis and evaluation of alternative substrates for arginase.
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 alpha-carboxyl group facilitates substrate binding, and the alpha-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.
Han
Shoufa
S
Department of Chemistry, University of Toledo, Toledo, OH 43606, USA.
Moore
Roger A
RA
Viola
Ronald E
RE
D000818:Animals; D001119:Arginase / Q000737:chemistry / Q000378:metabolism*; D001120:Arginine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D001565:Benzoates / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D006146:Guanidines / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009578:Nitrobenzenes / Q000737:chemistry / Q000378:metabolism; D009596:Nitrophenols / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D013890:Thiourea / Q000737:chemistry
C421260:1-nitro-3-guanidinobenzene; D001565:Benzoates; D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D006146:Guanidines; D009578:Nitrobenzenes; D009596:Nitrophenols; C461241:thioarginine; D001120:Arginine; D001119:Arginase; D013890:Thiourea