Inhibition of human arginase I by substrate and product analogues

Abstract

Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. We demonstrate that N-hydroxy-L-arginine (NOHA) binds to this enzyme with K(d)=3.6 microM, and nor-N-hydroxy-L-arginine (nor-NOHA) binds with K(d)=517 nM (surface plasmon resonance) or K(d) approximately 50 nM (isothermal titration calorimetry). Crystals of human arginase I complexed with NOHA and nor-NOHA afford 2.04 and 1.55 A resolution structures, respectively, which are significantly improved in comparison with previously-determined structures of the corresponding complexes with rat arginase I. Higher resolution structures clarify the binding interactions of the inhibitors. Finally, the crystal structure of the complex with L-lysine (K(d)=13 microM) is reported at 1.90 A resolution. This structure confirms the importance of hydrogen bond interactions with inhibitor alpha-carboxylate and alpha-amino groups as key specificity determinants of amino acid recognition in the arginase active site.

Figure 1

(a) Sensorgram showing the interaction of nor-NOHA with human arginase I, yielding K_d = 517 nM. (b) Sensorgram showing the interaction of NOHA with human arginase I, yielding K_d = 3.6 μM. (c) Plot of equilibrium concentrations (R_eq) determined by surface plasmon resonance for the complexation of human arginase I with L-lysine yields K_d = 13.1 μM; binding kinetics are too rapid to facilitate direct determination of association and dissociation rate constants (inset). (d) Isothermal titration calorimetry of nor-NOHA binding to human arginase I. Shown are the raw data obtained by titration of 0.036 mM arginase with 30 × 5 μL injections of 1.5 mM nor-NOHA (top). The area of each peak is integrated and plotted against [nor-NOHA]/[arginase] (bottom); nonlinear least-squares fitting of the data to a two-site model (solid line) yields K_d values of 51 nM and 47 nM.

Figure 2

(a) Stereoview of a simulated annealing gradient omit map contoured at 3.3σ, in which nor-NOHA bound in the active site of human arginase I (monomer A) was omitted from the structure factor calculation. Manganese coordination and hydrogen bond interactions are indicated by red and green dashed lines, respectively. Atom color codes: carbon (yellow), oxygen (red), nitrogen (blue), manganese (violet). (b) Superposition of the human arginase I-nor-NOHA complex (color coded as in (a)) and the rat arginase I-nor-NOHA complex (blue) [21].

Figure 3

(a) Stereoview of a simulated annealing gradient omit map contoured at 2.6σ, in which NOHA bound in the active site of human arginase I (monomer A) was omitted from the structure factor calculation. Manganese coordination and hydrogen bond interactions are indicated by red and green dashed lines, respectively; atoms are color coded as in Figure 2. (b) Superposition of the human arginase I-NOHA complex (color coded as in (a)) and the rat arginase I-NOHA complex (blue) [21].

Figure 4

(a) Stereoview of a simulated annealing gradient omit map contoured at 2.8σ, in which L-lysine bound in the active site of human arginase I (monomer A) was omitted from the structure factor calculation. Manganese coordination and hydrogen bond interactions are indicated by red and green dashed lines, respectively; atoms are color coded as in Figure 2. (b) Superposition of the human arginase I-L-lysine complex (color coded as in (a)) and the B. caldovelox arginase-L-lysine complex (blue) [36].

Figure 5

Summary of intermolecular interactions in the human arginase I-nor-NOHA complex. Metal coordination and hydrogen bond interactions are indicated by red and green dashed lines, respectively. We speculate that the zwitterionic form of the N-hydroxyguanidine moiety may bind as illustrated.

Figure 6

Superposition of human arginase I structures: native (red; the metal-bridging hydroxide ion is a smaller red sphere), nor-NOHA complex (blue), and NOHA complex (green). Active site Mn²⁺ ions are large spheres color coded according to their respective structures. Note that the hydroxyl oxygen of nor-NOHA lies closer to the position of the metal-bridging hydroxide ion of the native enzyme.