Mónica Salas

Interaction of arginase with metal ions: studies of the enzyme from human liver and comparison with other arginases

As determined by atomic absorption, fully activated human liver arginase contained 1.1 +/- 0.1 Mn2+/subunit. Upon dissociation to inactive subunits (< 0.01 Mn2+/subunit), there was decreased intensity and a red shift in the tryptophan fluorescence emission spectra of the enzyme, and the resulting species were markedly sensitive to thermal and proteolytic inactivation by trypsin. Arginine and lysine specifically protected the subunits from heat inactivation. Subunit activation by Mn2+ followed hyperbolic kinetics (Kd = 0.08 +/- 0.01 microM). In addition to Mn2+, Ni2+ and Co2+ converted inactive subunits into active monomers, and favoured their association to the oligomeric state of the enzyme (M(r) = 120,000 +/- 2000). The replacement of Mn2+ by Ni2+ or Co2+ resulted in significant changes in Vmax without any change in the Km values for the substrates (arginine or canavanine) or the Ki value for lysine inhibition. The results support our previous suggestion (Carvajal et al., 1994) that Mn2+ is not essential for substrate binding to arginase, and substantiates the conclusion that species differences may exist in the interaction of arginase with metal ions.

Manganese-dependent inhibition of human liver arginase by borate.

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.

Properties of an arginase from the cotyledons of Phaseolus vulgaris

Abstract In the cotyledons of Phaseolus vulgaris , the arginase activity reached a maximum at about 2–3 days of seed germination; thereafter, it began to decrease. The enzyme was partially purified and characterized. Canavanine was not a substrate and the K m values for arginine were 45 ± 2 mM at the optimum pH of 9.6 and 93 ± 5 mM at pH 7.5. Several metal ions activated the enzyme to a different degree (Mn 2+ > Co 2+ > Ni 2+ > Cd 2+ ), but the K m and K i values were the same for all the metal activated enzymes. Reactivation of fully inactivated enzyme by Mn 2+ followed hyperbolic kinetics with a K d value of 0.47 ± 0.05 μM.

Insights into the interaction of human liver arginase with tightly and weakly bound manganese ions by chemical modification and site-directed mutagenesis studies

Diethyl pyrocarbonate (DEPC) caused a loss in the ability of inactive subunits of wild-type and H141F mutant human liver arginase (EC 3.5.3.1) to be reactivated by Mn(2+). The effect was reversed by hydroxylamine and involved a residue with a pK(a) of 6.5+/-0.1. Half activation with Mn(2+) was sufficient for total resistance of H141F and full activation was not impeded by a previous incubation of the half-active species with DEPC. The H101N and H126N mutants expressed 60 and 82% of the wild-type activity, respectively, without changes in K(m) for arginine or K(i) for lysine inhibition. After dialysis against EDTA, H126N was inactive in the absence of added Mn(2+) and contained <0.1 Mn(2+)/subunit, whereas H101N was half active and contained 1.2+/-0.1 Mn(2+)/subunit. Results support the concept that a weakly bound metal ion is needed only for conversion of active species to a more active active state.

Implications of Resveratrol on Glucose Uptake and Metabolism

Resveratrol—a polyphenol of natural origin—has been the object of massive research in the past decade because of its potential use in cancer therapy. However, resveratrol has shown an extensive range of cellular targets and effects, which hinders the use of the molecule for medical applications including cancer and type 2 diabetes. Here, we review the latest advances in understanding how resveratrol modulates glucose uptake, regulates cellular metabolism, and how this may be useful to improve current therapies. We discuss challenges and findings regarding the inhibition of glucose uptake by resveratrol and other polyphenols of similar chemical structure. We review alternatives that can be exploited to improve cancer therapies, including the use of other polyphenols, or the combination of resveratrol with other molecules and their impact on glucose homeostasis in cancer and diabetes.

A critical histidine residue in arginase from Phaseolus vulgaris

Abstract Arginase from Phaseolus vulgaris was inactivated by diethyl pyrocarbonate (DEPC). The bimolecular rate constant for inactivation by DEPC was 1300 M −1 min −1 and the reaction order with respect to DEPC concentration was ca 1. The inactivation followed a titration curve for a residue with a p K a of 6.7±0.1 at 25° and the enzymatic activity was completely restored by hydroxylamine. Results are taken as evidence for a critical, but not essential, histidine residue in the enzyme.

Chemical Modification of Genypterus maculatus Arginase by Woodward's Reagent K and Diethyl Pyrocarbonate: Evidence for an Essential Carboxylate and a Nonessential, Albeit Important Histidine Residue

Abstract Liver arginase from Genypterus maculatus was completely inactivated by Woodwards reagent K (WRK) and partially inactivated by diethyl pyrocarbonate (DEPC). Borate protected against inactivation by DEPC and caused a nontotal inhibition of the enzyme. Inactivation by WRK (second order rate constant, 26.3 M−1 min−1) was associated to the chemical modification of a single residue with a pKa value of 6.5 at 25°C. Inactivation by DEPC (second order rate constant, 336 M−1 min−1) involved a single residue with a pKa of 6.8 at 25°C and it was reversed by hydroxylamine. A carboxylate residue associated to the action of a metal-bound hydroxyl as a nucleophile, and a histidine residue with a nonessential role in the catalytic mechanism of arginase, are considered.