DOI: 10.1002/chem.200802252 Determination of the Catalytic Pathway of a Manganese Arginase Enzyme Through Density Functional Investigation Monica Leopoldini, Nino Russo,* and Marirosa Toscano [a] Dedicated to the Centenary of the Italian Chemical Society Introduction Arginase (l-arginine amidinohydrolase, EC 3.5.3.1) is a ho- motrimeric enzyme that catalyzes the hydrolysis of l-argi- nine to l-ornithine and urea. [1–3] Two genetically distinct iso- zymes have evolved with differing tissue distributions and subcellular locations in mammals. [4, 5] Arginase I is found in the liver, where it catalyzes the final cytosolic step of the urea cycle and is responsible for the generation of  10 kg of urea per year by the average human adult. [2, 3] Urea is the principal metabolite for removal of nitrogen as a non-toxic product formed during amino acid metabolism in mammals. Arginase II is a mitochondrial enzyme not involved in the urea cycle, which is present in numerous tissues, for exam- ple, kidney, brain, skeletal muscle, and liver. [6, 7] Experiments suggest that arginase II operates in l-arginine homeostasis by regulating l-arginine concentrations in those tissues where it represents the substrate for other biosynthetic reac- tions, such as nitric oxide (NO) biosynthesis. [8] l-Ornithine serves as a biosynthetic precursor for the pro- duction of l-proline and polyamines, such as putrescine, spermina (in eucaryotes), and spermidine (in procaryotes), whereby it is decarboxylated under catalysis by ornithine de- carboxylase. [9–13] These polyamines are found in high concen- trations in actively growing cells, where they act as growth factors. Polyamines also serve to stabilize membrane struc- tures of bacteria, as well as the structures of ribosomes and some viruses. [12, 13] Macrophages use l-arginine to synthesize nitric oxide (NO) and polyamines through inducible NO synthase (iNOS) and arginase, respectively. NO contributes to the tu- moricidal activity of macrophages, whereas polyamines may promote the growth of cancer cells. [9–13] Experimental find- ings [14, 15] demonstrate that overexpression of arginase in these cells enhances the production of polyamines, the es- sential nutrients required for the proliferation, differentia- tion, and neoplastic transformation of mammalian cells. Thus, it is likely that macrophages are capable of promoting tumor cell proliferation through the arginase pathway. The existence of a large volume of literature suggests that better knowledge of the arginase catalytic mechanism may provide far-reaching insights from the chemical, biological, and medicinal points of view. Abstract: The catalytic mechanism of dimanganese-containing arginase enzyme has been investigated by DFT calculations. Two exchange-correlation functionals, B3 LYP and MPWB1 K, have been used to construct the poten- tial energy profiles for the hydrolysis of an arginine substrate performed by an arginase active site model system. Two reaction mechanisms have been investi- gated, one involving a water molecule (mechanism 1) and the other involving a hydroxide ion (mechanism 2) as nu- cleophilic agent. Results obtained in the gas phase and in the protein envi- ronment have indicated that mecha- nism 1 involving a water molecule en- tails structural features as well as an activation energy for the rate-deter- mining step that are inconsistent with experimental data available for the ar- ginase enzyme. On the other hand, when a hydroxide ion is present at the Mn2 site, a lower activation energy and a structural arrangement closer to the experimental indication are obtained. Keywords: arginase · catalytic mechanism · density functional cal- culations · metalloenzymes · transi- tion states [a] Dr. M. Leopoldini, Prof. N. Russo, Prof. M. Toscano Dipartimento di Chimica and Centro di Calcolo ad Alte Prestazioni per Elaborazioni Parallele e Distribuite-Centro d’Eccellenza MIUR Universita’ della Calabria 87030 Arcavacata di Rende (CS) (Italy) Fax: (+ 39)0984-493390 E-mail : nrusso@unical.it  2009 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Chem. Eur. J. 2009, 15, 8026 – 8036 8026