PMC

Diagram of selected substrates and inhibitors of NOS and DDAH-1.

Reversible covalent inhibition of DDAH-1 by L-IPO (13). The the non-covalent complex (20) is expected to be in rapid equilibrium with the (R)-tetrahedral complex (21).

Nitric oxide biosynthesis is promoted by the enzymic activities of both NO synthase (NOS) and DDAH. DDAH catabolizes endogenous methylarginines and relieves their inhibition of NOS.

The active site of DDAH-1 in complex with L-IPO (13) and omit map density. Atoms colored in cyan, blue, red and yellow for carbon, nitrogen, oxygen and sulfur, respectively. Active-site residues and L-IPO are labeled. The F_o-F_c electron density map, with L-IPO and the C274 Sγ atom not included in the calculated structure factors, is shown at 3 σ in blue.

Lineweaver-Burk plot of DDAH-1 inhibition by L-IPO (). L-IPO is included in assay mixtures at 0 (●), 62.5 (▼), 125 (▲), 250 (□) and 500 (■) μM at pH 7.27, 25 °C. Intersecting fits at 1/V_max indicates competitive inhibition against the SMTC substrate. A K_i value of 50 ± 4 μM is determined as described in Experimental Procedures.

Analytical sedimentation equilibrium ultracentrifugation of DDAH-1. Symbols represent an overlay of data collected during the last nine scans, indicative that equilibrium had been reached. The solid line represents the global simultaneous fit for a single ideal species model using Ultrascan. The fitted M_w is 34,530 Da (theoretical monomer M_w = 33,558 Da), variance = 8.7e^-5. Conditions: DDAH-1 (1 mg/ mL), NaH₂PO₄ (20 mM), NaCl (100 mM), pH 7.0 at 25 °C with a rotor speed of 20,000 rpm.

Alternative loop conformations observed in DDAH-1. Structural variance in residues 167 – 173 is observed depending on the bound inhibitor. Protein backbone is shown as a ribbon representation, bound inhibitors as ball and stick representations, and selected residues (Arg 145, Leu172, His 173, Cys 274) in stick form. Apo protein is in white, and the l-citrulline (4), L-IPO (13) and L-257 (16) complexed structures in green, light blue and orange, respectively. The figure is constructed using coordinates from this work and from protein data bank accession codes 2JAI and 2JAJ ().

Comparison of DDAH-1 and NOS active sites. The left panel shows a superimposition of human DDAH-1 with bound L-IPO (13) (green) and L-257 (16) (pink). The right panel shows a superimposition of bovine eNOS bound by L-NIO (12) (green) and rat nNOS bound by N^ω-propyl-l-arginine (pink), a compound of comparable length to N⁵-(1-iminopentyl)-l-ornithine (14). Inhibitors are shown in ball and stick format and colored by heteroatom as described earlier. The heme groups of NOS are shown in stick models. Surface features (light blue) are shown for the L-257-DDAH-1 complex and the N^ω-propyl-l-arginine-nNOS complex to highlight the active-site cavity shape. Figures are constructed using coordinates from this work and protein data bank accession codes 2JAJ, 1ED6 and 1MMV (, , ).