Active Site Of Arginase Research Articles

Design and Synthesis of Acyclic Boronic Acid Arginase Inhibitors.

Arginase has long been a target of interest in immuno-oncology, but discovering an orally bioavailable inhibitor is severely constrained by the requisite boronic acid pharmacophore. We began our drug discovery campaign by building off the β-position of the literature inhibitor ABH (1). A divergent synthesis with an Ireland-Claisen rearrangement as the key step allowed access to numerous compounds, some of which we crystallized in the active site of arginase 2. We subsequently used structure-based drug design to further improve the potency of this series, ultimately achieving an inhibitor with an IC50 value of 12 nM. Many compounds in this series were designed to behave as prodrugs, releasing their payload with up to 4-fold improved oral exposure relative to the parent. Subtle stereochemical differences between these various inhibitors and prodrugs had substantial effects on potency and pharmacokinetics.

Metal-induced change in catalytic loop positioning in Helicobacter pylori arginase alters catalytic function.

Arginase is a bimetallic enzyme that utilizes mainly Mn2+ or Co2+ for catalytic function. In human homolog, the substitution of Mn2+ with Co2+ significantly reduces the Km value without affecting the kcat. However, in the Helicobacter pylori counterpart (important for pathogenesis), the kcat increases nearly 4-fold with Co2+ ions both in the recombinant holoenzyme and arginase isolated from H. pylori grown with Co2+ or Mn2+. This suggests that the active site of arginase in the two homologs is modulated differently by these two metal ions. To investigate the underlying mechanism for metal-induced difference in catalytic activity in the H. pylori enzyme, we used biochemical, biophysical and microsecond molecular dynamics simulations studies. The study shows that the difference in binding affinity of Co2+ and Mn2+ ions with the protein is linked to a different positioning of a loop (-122HTAYDSDSKHIHG134-) that contains a conserved catalytic His133. Consequently, the proximity of His133 and conserved Glu281 is varied. We found that the Glu281-His133 interaction is crucial for catalytic function and was previously unexplored in other homologs. We suggest that the proximity difference between these two residues in the Co2+- and Mn2+-proteins alters the proportion of protonated His133 via variation in its pKa. This affects the efficiency of proton transfer - an essential step of l-arginine hydrolysis reaction catalyzed by arginase and thus activity. Unlike in human arginase, the flexibility of the above segment observed in H. pylori homolog suggests that this region in the H. pylori enzyme may be explored to design its specific inhibitors.

Impact of Substrate Protonation and Tautomerization States on Interactions with the Active Site of Arginase I

Human arginase is a binuclear manganese metalloenzyme that participates in the urea cycle. Arginase catalyzes the hydrolysis of L-arginine into L-ornithine and urea and is linked to several disorders such as asthma and cancer. Currently, the protonation and tautomerization state of the substrate when bound to the active site, which contains two manganese ions, is not known. Knowledge of the charge-dependent behavior of arginine in the arginase I environment would be of utility toward understanding the catalytic mechanism and designing inhibitors of this enzyme. The arginine(+/0) species, including all possible neutral tautomers, were modeled using an aminoimidazole analog as template. All-atom molecular dynamics simulations were then performed on each of the charged and neutral species. In addition, a hydroxide ion was included in selected simulations to test its importance. Results show that the positively charged state of arginine is stable in the active site of arginase I, with that stabilization facilitated by the presence of hydroxide. Glu277 is indicated to play a role in stabilizing arginine in the active site and facilitating its ability to assume a catalytically competent conformation in the presence of hydroxide. The reported interactions and modeled arginine-bound arginase I structures can be used as a tool for structure-based inhibitor design, as experimental data on the structure of the substrate-enzyme complex is lacking.

The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase

Polyamine biosynthesis enzymes are promising drug targets for the treatment of leishmaniasis, Chagas’ disease and African sleeping sickness. Arginase, which is a metallohydrolase, is the first enzyme involved in polyamine biosynthesis and converts arginine into ornithine and urea. Ornithine is used in the polyamine pathway that is essential for cell proliferation and ROS detoxification by trypanothione. The flavonols quercetin and quercitrin have been described as antitrypanosomal and antileishmanial compounds, and their ability to inhibit arginase was tested in this work. We characterized the inhibition of recombinant arginase from Leishmania (Leishmania) amazonensis by quercetin, quercitrin and isoquercitrin. The IC50 values for quercetin, quercitrin and isoquercitrin were estimated to be 3.8, 10 and 4.3μM, respectively. Quercetin is a mixed inhibitor, whereas quercitrin and isoquercitrin are uncompetitive inhibitors of L. (L.) amazonensis arginase. Quercetin interacts with the substrate l-arginine and the cofactor Mn2+ at pH 9.6, whereas quercitrin and isoquercitrin do not interact with the enzyme’s cofactor or substrate. Docking analysis of these flavonols suggests that the cathecol group of the three compounds interact with Asp129, which is involved in metal bridge formation for the cofactors MnA2+ and MnB2+ in the active site of arginase. These results help to elucidate the mechanism of action of leishmanicidal flavonols and offer new perspectives for drug design against Leishmania infection based on interactions between arginase and flavones.

Inhibition of the Arginase Catalyzed Hydrolysis of L-Arginine by La 3+

The inhibitive role of lanthanum ion on the arginase catalyzed hydrolysis of L-arginine was studied by microcalorimetry in a 40 mmol/L sodium barbiturate-hydrochloric acid buffer solution at 37 ℃ and pH of 9.4.The results indicated La3+ has remarkable inhibition effect on the catalytic activity of arginase and the inhibitory type belongs to the reversible non-competitive inhibition with the inhibition constant of 2.74×10-4 mol/L.On the basis of the inhibition type and ion radius,we suggested that the binding site of La3+ to arginase is far from the active site of arginase.

Theoretical studies on the structural and magnetic property of arginase active site

The chemical species of solvent molecule (WB) between two manganese ions in the active site of arginase is investigated based on the structural and magnetic property. The calculated magnetic coupling constant (J ab ) values at UBH&HLYP level of theory are − 1.9 cm− 1 for WB = H2O and − 5.7 cm− 1 for WB = OH− , respectively. In comparison with the experimental value (J ab = ∼ − 2.0 cm− 1), H2O is the most likely candidate to WB in the initial structure. Natural orbital analyses reveal that WB plays an important role for keeping the geometry of the Mn–O(WB)–Mn core rigid rather than mediating superexchange interaction. The result indicates that WB does not have to shift to one Mn ion as an initiation of the hydrolysis reaction.

Probing the Specificity Determinants of Amino Acid Recognition by Arginase

Arginase is a binuclear manganese metalloenzyme that serves as a therapeutic target for the treatment of asthma, erectile dysfunction, and atherosclerosis. In order to better understand the molecular basis of inhibitor affinity, we have employed site-directed mutagenesis, enzyme kinetics, and X-ray crystallography to probe the molecular recognition of the amino acid moiety (i.e., the alpha-amino and alpha-carboxylate groups) of substrate l-arginine and inhibitors in the active site of arginase I. Specifically, we focus on (1) a water-mediated hydrogen bond between the substrate alpha-carboxylate and T135, (2) a direct hydrogen bond between the substrate alpha-carboxylate and N130, and (3) a direct charged hydrogen bond between the substrate alpha-amino group and D183. Amino acid substitutions for T135, N130, and D183 generally compromise substrate affinity as reflected by increased K(M) values but have less pronounced effects on catalytic function as reflected by minimal variations of k(cat). As with substrate K(M) values, inhibitor K(d) values increase for binding to enzyme mutants and suggest that the relative contribution of intermolecular interactions to amino acid affinity in the arginase active site is water-mediated hydrogen bond < direct hydrogen bond < direct charged hydrogen bond. Structural comparisons of arginase with the related binuclear manganese metalloenzymes agmatinase and proclavaminic acid amidinohydrolase suggest that the evolution of substrate recognition in the arginase fold occurs by mutation of residues contained in specificity loops flanking the mouth of the active site (especially loops 4 and 5), thereby allowing diverse guanidinium substrates to be accommodated for catalysis.

Dynamical Flexibility and Proton Transfer in the Arginase Active Site Probed by ab Initio Molecular Dynamics

We have used ab initio molecular dynamics (AIMD) to investigate the dynamical flexibility of the bridged binuclear structural motif in the active site of arginase. Dynamical transformations play a crucial role in catalysis. We have provided direct insight into the motions of the first-shell ligands with emphasis on the chelating and bridging carboxylates. In the case of the terminal Asp234 residue we observe changes in the binding mode (carboxylate shifts). AIMD dynamics of sufficient duration has allowed us to observe proton transfer from the bridging nucleophile to the catalytically essential Asp 128 residue and to map the underlying free energy surface in terms of simple reaction coordinates, such as the oxygen-oxygen distance Ro-o and the asymmetric stretch delta. This has provided valuable insight into the nature of the last step of the catalytic cycle. In addition, constrained molecular dynamics permitted us to compare the deprotonation free energy of the bridging nucleophile in the case of native versus metal-depleted arginase.

Thermokinetic studies of the irreversible inhibition of single-substrate, enzyme-catalyzed reactions

A thermokinetic ratio method for the irreversible inhibition of single-substrate enzymecatalyzed reactions is proposed in this paper. By analyzing a measured curve this method can be used to calculate the apparent rate constant of the inhibited reaction without letting the reaction go to completion. Using the LKB-2107 batch microcalorimeter, the arginase-catalyzed hydrolysis of L-arginine in the presence of p-chloromercuribenzoate (PCMB) has been studied and PCMB established as an irreversible mixed inhibitor. The second-order rate constants for inhibition of arginase by PCMB in the absence and presence of L-arginine have been determined by this ratio method to be k EI = 94.4 M − s −1 and k ESI = 35.2 M −1 s −1, respectively, at 298.15 K. Chemical modification with PCMB indicates that arginase contains three reactive cysteinyl residues at most but these residues are not present at the active site of arginase.