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^,

Abstract

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.

Crystal structure of human arginase I at 1.29-Å resolution and exploration of inhibition in the immune response

“…Diffraction intensities measured from these crystals exhibited symmetry consistent with the apparent space group P6 (unit cell parameters a ϭ b ϭ 91.4 Å and c ϭ 69.6 Å). These crystals were isomorphous with those obtained of the rat arginase I-ABH and -BEC complexes (23,24). Data reduction was achieved with DENZO and SCALEPACK (28).…”

“…Human arginase I activity was measured by using the radioactive assay of Rüegg and Russell (34), as described, for the study of rat arginase I activity (23). Inhibitor affinity was measured by using isothermal titration calorimetry with an MCS isothermal titration calorimeter (Microcal Software, Northampton, MA), and a procedure identical to that described for the assay of rat arginase I-inhibitor affinity (23).…”

“…The structure of rat arginase I has been exceptionally useful for understanding inhibitor structureaffinity relationships (23)(24)(25). However, given the potential pharmaceutical importance of the human enzyme, an experimentally determined structure is critical for guiding the structure-based design of potent and isozyme-selective inhibitors.…”

Endothelial dysfunction in diabetic erectile dysfunction

“…22 Recent in vitro studies proposed that oxidized LDL in particular is able to decrease local endothelial L-arginine uptake, ultimately leading to both local depletion of L-arginine and eNOS uncoupling. 23 Reduced bioavailable levels of L-arginine for [24][25][26] Inhibition of arginase in the diabetic human penis shifts the availability of L-arginine to NOS and enhances NOS activity and cavernosum smooth muscle relaxation. 25 Inactivation of eNOS cofactor tetrahydrobiopterin (BH 4 ) also reduces eNOS activity.…”

Ligand-gated Ion Channels

“…(Tenu et al, 1999), S-(2-boronoethyl)-L-cysteine (Colleluori and Ash, 2001;Kim et al, 2001) and 2(S)-amino-6-boronohexanoic acid (Baggio et al, 1999;Colleluori and Ash, 2001). Other names Dimethylarginine dimethylaminohydrolase 1, DDAHI, dimethylargininase-1 Dimethylarginine dimethylaminohydrolase 2, DDAHII, dimethylargininase-2, S-phase protein, protein G6a…”