pH-Sensitive control of arginase by Mn(II) ions at submicromolar concentrations

Kuhn, Nicholas J.; Talbot, J.; Ward, Simon

doi:10.1016/0003-9861(91)90031-d

Cited by 50 publications

(29 citation statements)

References 22 publications

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“…Given the similar pH rate profiles of AMPP (27) and arginase (28), it is most likely that the ligand that bridges the two Mn 2ϩ ions in the active form of the enzymes is a hydroxide ion rather than a water molecule (18). It has been suggested that in arginase this hydroxide acts as the nucleophile (18), and our structures support this hypothesis for AMPP.…”

Section: Discussionsupporting

confidence: 74%

Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli

Wilce

Bond

Dixon

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

185

210

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The structure of the proline-specific aminopeptidase (EC 3.4.11.9) from Escherichia coli has been solved and refined for crystals of the native enzyme at a 2.0-Å resolution, for a dipeptide-inhibited complex at 2.3-Å resolution, and for a low-pH inactive form at 2.7-Å resolution. The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions. The monomer folds into two domains. The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro. The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits. The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme). The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center.Proline-specific peptidases have been described in a wide variety of organisms and specifically cleave either the amide bond after a proline residue or the imide bond that precedes it (1). Proline aminopeptidases (EC 3.4.11.9) specifically release the N-terminal residue from a peptide where the penultimate residue is proline. These enzymes have significant sequence homology with (i) some other amino peptidases (e.g., methionine aminopeptidase, AMPM) and (ii) the Xaa-Pro dipeptidases, prolidases, that specifically cleave Xaa-Pro dipeptides. All these enzymes are activated in the presence of divalent metal ions, though the biologically active metal is not always certain. In vitro the prolyl peptidases are most active in the presence of Mn 2ϩ . Structural comparisons between AMPM and creatine amidinohydrolase (creatinase) and sequence comparisons between these proteins and aminopeptidase P (AMPP) and prolidase have suggested that the catalytic domains of all four enzymes have a common fold (2). This work presents the crystal structure of AMPP, the product of the pepP gene in Escherichia coli. The structures of a complex of AMPP with a dipeptide inhibitor and a low-pH inactive form are used to devise a plausible mechanism for peptide hydrolysis. EXPERIMENTAL PROCEDURESOverexpression and Purification of AMPP. AMPP was originally isolated and characterized in E. coli (3) and was subsequently cloned and overexpressed (4). The details of the overexpression and purification of AMPP used in this work will be published elsewhere. Briefly, AMPP was overexpressed in the E. coli AN1459͞pPL670. Lysed cells were subject to (NH 4 ) 2 SO 4 precipitation at 4°C followed by centrifugation. The pellet was resuspended and applied to a DEAE-Fractogel column (Merck). The AMPP-containing fraction was then passed over a ceramic hydroxyapatite column (Bio-Rad). This simple purifi...

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Section: Discussionsupporting

confidence: 74%

Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli

Wilce

Bond

Dixon

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

185

210

View full text Add to dashboard Cite

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“…The pK a of this catalytic nucleophile or of a catalylic base in equilibrium with it has been determined to be about 7.9-8.0 [50,51]. This pK a is 2-2.5 units lower than that found in mononuclear Mn(II) complexes such as aqua Mn(II) in solution (Eq.…”

Section: Discussionmentioning

confidence: 98%

Catalysis on dinuclear Mn(II) centers: hydrolytic and redox activities of rat liver arginase

et al. 1997

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Rat liver arginase contains a dinuclear Mn 2 (II,II) center in each subunit having EPR properties similar to those observed in Mn-catalases. The principal physiologic role of arginase is catalyzing the hydrolytic cleavage of L-arginine to produce L-ornithine and urea. Here we demonstrate that arginase catalyzes the disproportionation of hydrogen peroxide by a redox mechanism analogous to Mn-catalases, but at rates that are 10 P5 to 10 P6 of k cat for the Mn-catalases, and also exhibits peroxidase activity. The dinuclear Mn 2 (II,II) center is essential for maximal catalase activity, since both the H101N and H126N mutant arginases containing only one Mn(II)/subunit have catalase activities that are ~3% of that for the wild-type enzyme. Like the Mn-catalases, the catalase activity of arginase is not inhibited by millimolar concentrations of CN P , the most potent inhibitor of heme catalases, or by EDTA, a chelator of free metal ions. The catalase activity of arginase is not significantly inhibited by Cl P or F P , in contrast to Mn-catalases, while potent inhibitors of the hydrolytic activity are also effective inhibitors of the catalase activity. These results suggest that lower affinity of hydrogen peroxide to the active site of arginase contributes to the lower catalase activity. EPR spectroscopy reveals that potent inhibitors of the hydrolytic reaction, including N v -hydroxy-L-arginine, Llysine, and L-valine, decouple the electronic interaction between the Mn 2c ions, most probably by removing a m-bridging ligand or by increasing the intermanganese separation. The capacity for arginase to deliver a hydroxide ion to hydrolyze the L-arginine substrate is suggested to arise from a "dinuclear effect", wherein the two metal ions contribute more or less equivalently in deprotonation of metal-bound water molecule. Structure-reactivity analyses of these reactions will provide insights into the factors that control redox versus hydrolytic function in dimanganese clusters. Key words Arginase 7 Manganoenzymes 7 Catalase 7 PeroxidaseAbbreviations CAPS 3-(cyclohexylamino)-1-propanesulfonic acid 7 CAPSO 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid 7 EPR electron paramagnetic resonance 7 ENDOR electron-nuclear double resonance 7 EXAFS extended X-ray absorption fine structure 7 HEPES N-(2-hydroxyethyl)-piperazine-Nb-(2-ethanesulfonic acid) 7 MES 2-(N-morpholino)ethanesulfonic acid 7 TRIS tris(hydroxymethyl)-aminomethane 7 ZFS zero-field splitting

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“…Further support for mechanism 2 is provided by the experimental finding [58] that the optimum pH range for arginase catalysis is between 9 and 9.5.…”

mentioning

confidence: 85%

Determination of the Catalytic Pathway of a Manganese Arginase Enzyme Through Density Functional Investigation

Leopoldini

Russo

Toscano

2009

Chemistry A European J

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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 potential energy profiles for the hydrolysis of an arginine substrate performed by an arginase active site model system. Two reaction mechanisms have been investigated, one involving a water molecule (mechanism 1) and the other involving a hydroxide ion (mechanism 2) as nucleophilic agent. Results obtained in the gas phase and in the protein environment have indicated that mechanism 1 involving a water molecule entails structural features as well as an activation energy for the rate-determining step that are inconsistent with experimental data available for the arginase 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.

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pH-Sensitive control of arginase by Mn(II) ions at submicromolar concentrations

Cited by 50 publications

References 22 publications

Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli

Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli

Catalysis on dinuclear Mn(II) centers: hydrolytic and redox activities of rat liver arginase

Determination of the Catalytic Pathway of a Manganese Arginase Enzyme Through Density Functional Investigation

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