Probing erectile function: S-(2-boronoethyl)-L-cysteine binds to arginase as a transition state analogue and enhances smooth muscle relaxation in human penile corpus cavernosum

The boronic acid-based arginine analogue S-(2-boronoethyl)-L-cysteine (BEC) has been synthesized and assayed as a slow-binding competitive inhibitor o f the binuclear manganese metalloenzyme arginase. Kinetic measurements indi cate a K-I value of 0.4-0.6 muM, which is in reasonable agreement with the dissociation constant of 2.22 muM measured by isothermal titration calorime try. The X-ray crystal structure of the arginase-BEC complex has been deter mined at 2.3 Angstrom resolution from crystals perfectly twinned by hemihed ry. The structure of the complex reveals that the boronic acid moiety under goes nucleophilic attack by metal-bridging hydroxide ion to yield a tetrahe dral boronate anion that bridges the binuclear manganese cluster, thereby m imicking the tetrahedral intermediate land its flanking transition states) in the arginine hydrolysis reaction. Accordingly, the binding mode of BEC i s consistent with the structure-based mechanism proposed for arginase as ou tlined in Cox et al. [Cox, J. D., Cama, E., Colleluori D. M., Pethe, S., Bo ucher, J. S., Mansuy, D., Ash, D. E., and Christianson, D. W. (2001) Bioche mistry 40, 2689-2701.]. Since BEC does not inhibit nitric oxide synthase, B EC serves as a valuable reagent to probe the physiological relationship bet ween arginase and nitric oxide (NO) synthase in regulating the NO-dependent smooth muscle relaxation in human penile corpus cavernosum tissue that is required for erection. Consequently, we demonstrate that arginase is presen t in human penile corpus cavernosum tissue, and that the arginase inhibitor BEC causes significant enhancement of NO-dependent smooth muscle relaxatio n in this tissue. Therefore, human penile arginase is a potential target fo r the treatment of sexual dysfunction in the male.