The boronic acid-based arginine analogue S-(2-boronoethyl)-L-cysteine (BEC) has been
synthesized and assayed as a slow-binding competitive inhibitor of the binuclear manganese
metalloenzyme arginase. Kinetic measurements indicate a K(I) value of 0.4-0.6 microM,
which is in reasonable agreement with the dissociation constant of 2.22 microM measured
by isothermal titration calorimetry. The X-ray crystal structure of the arginase-BEC
complex has been determined at 2.3 A resolution from crystals perfectly twinned by
hemihedry. The structure of the complex reveals that the boronic acid moiety undergoes
nucleophilic attack by metal-bridging hydroxide ion to yield a tetrahedral boronate
anion that bridges the binuclear manganese cluster, thereby mimicking the tetrahedral
intermediate (and its flanking transition states) in the arginine hydrolysis reaction.
Accordingly, the binding mode of BEC is consistent with the structure-based mechanism
proposed for arginase as outlined in Cox et al. [Cox, J. D., Cama, E., Colleluori
D. M., Pethe, S., Boucher, J. S., Mansuy, D., Ash, D. E., and Christianson, D. W.
(2001) Biochemistry 40, 2689-2701.]. Since BEC does not inhibit nitric oxide synthase,
BEC serves as a valuable reagent to probe the physiological relationship between 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 present in human penile corpus cavernosum tissue,
and that the arginase inhibitor BEC causes significant enhancement of NO-dependent
smooth muscle relaxation in this tissue. Therefore, human penile arginase is a potential
target for the treatment of sexual dysfunction in the male.