Arginase is a binuclear Mn(2+) metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. X-ray crystal structures of arginase complexed to substrate analogues N(omega)-hydroxy-L-arginine and N(omega)-hydroxy-nor-L-arginine, as well as the products L-ornithine and urea, complete a set of structural "snapshots" along the reaction coordinate of arginase catalysis when interpreted along with the X-ray crystal structure of the arginase-transition-state analogue complex described in Kim et al. [Kim, N. N., Cox, J. D., Baggio, R. F., Emig, F. A., Mistry, S., Harper, S. L., Speicher, D. W., Morris, Jr., S. M., Ash, D. E., Traish, A. M., and Christianson, D. W. (2001) Biochemistry 40, 2678-2688]. Taken together, these structures render important insight on the structural determinants of tight binding inhibitors. Furthermore, we demonstrate for the first time the structural mechanistic link between arginase and NO synthase through their respective complexes with N(omega)-hydroxy-L-arginine. That N(omega)-hydroxy-L-arginine is a catalytic intermediate for NO synthase and an inhibitor of arginase reflects the reciprocal metabolic relationship between these two critical enzymes of L-arginine catabolism.
1 Cholinergic airway constriction is functionally antagonized by agonist-induced constitutive nitric oxide synthase (cNOS)-derived nitric oxide (NO). Since cNOS and arginase, which hydrolyzes Larginine to L-ornithine and urea, use L-arginine as a common substrate, competition between both enzymes for the substrate could be involved in the regulation of cholinergic airway reactivity. Using a perfused guinea-pig tracheal tube preparation, we investigated the modulation of methacholineinduced airway constriction by the recently developed, potent and speci®c arginase inhibitor N ohydroxy-nor-L-arginine (nor-NOHA). 2 Intraluminal (IL) administration of nor-NOHA caused a concentration-dependent inhibition of the maximal e ect (E max ) in response to IL methacholine, which was maximal in the presence of 5 mM nor-NOHA (E max =31.2+1.6% of extraluminal (EL) 40 mM KCl-induced constriction versus 51.6+2.1% in controls, P50.001). In addition, the pEC 50 (7log 10 EC 50 ) was slightly but signi®cantly reduced in the presence of 5 mM nor-NOHA. 3 The inhibition of E max by 5 mM nor-NOHA was concentration-dependently reversed by the NOS inhibitor N o -nitro-L-arginine methyl ester (L-NAME), reaching an E max of 89.4+7.7% in the presence of 0.5 mM L-NAME (P50.01). A similar E max in the presence of 0.5 mM L-NAME was obtained in control preparations (85.2+9.7%, n.s.). 4 In the presence of excess of exogenously applied L-arginine (5 mM), 5 mM nor-NOHA was ine ective (E max =33.1+5.8 versus 31.1+7.5% in controls, n.s.). 5 The results indicate that endogenous arginase activity potentiates methacholine-induced airway constriction by inhibition of NO production, presumably by competition with cNOS for the common substrate, L-arginine. This ®nding may represent an important novel regulation mechanism of airway reactivity.
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