RESUMO
Native and wild-type recombinant human liver arginases (EC 3.5.3.1) were photoinactivated by Rose bengal, and protection was afforded by the competitive inhibitor l-lysine. The dissociation constant for the enzyme-protector complex was essentially equal to the corresponding K(i) value. Upon mutation of His141 by phenylalanine, the enzyme activity was reduced to 6-10% of wild-type activity, with no changes in K(m) for arginine or K(i) for l-lysine or l-ornithine. The subunit composition of active enzyme was not altered by mutation, but the mutant H141F was markedly more sensitive to trypsin inactivation and completely insensitive to inactivation by diethyl pyrocarbonate (DEPC) and photoinactivation. Species with histidine groups blocked with DEPC were also insensitive to photoinactivation. We conclude that His141, which is the target for both inactivating procedures, is not involved in substrate binding, but plays a critical, albeit not essential role in the hydrolysis of enzyme-bound substrate.
Assuntos
Arginase/metabolismo , Histidina/genética , Fígado/enzimologia , Arginase/antagonistas & inibidores , Arginase/genética , Arginase/efeitos da radiação , Arginina/metabolismo , Domínio Catalítico , Dietil Pirocarbonato/farmacologia , Humanos , Luz , Lisina/metabolismo , Mutagênese Sítio-Dirigida , Ornitina/metabolismo , Rosa Bengala/farmacologiaRESUMO
Agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was inactivated by diethyl pyrocarbonate (DEPC) and illumination in the presence of Rose bengal. Protection against photoinactivation was afforded by the product putrescine, and the dissociation constant of the enzyme-protector complex (12 mM) was essentially equal to the K(i) value for this compound acting as a competitive inhibitor of agmatine hydrolysis. Upon mutation of His163 by phenylalanine, the agmatinase activity was reduced to 3-5% of wild-type activity, without any change in K(m) for agmatine or K(i) for putrescine inhibition. The mutant was insensitive to DEPC and dye-sensitized inactivations. We conclude that His163 plays an important role in the catalytic function of agmatinase, but it is not directly involved in substrate binding.
Assuntos
Escherichia coli/enzimologia , Histidina/metabolismo , Ureo-Hidrolases/metabolismo , Catálise , Dietil Pirocarbonato/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Cinética , Mutagênese Sítio-Dirigida , Rosa Bengala/metabolismo , Especificidade por Substrato , Ureo-Hidrolases/antagonistas & inibidores , Ureo-Hidrolases/genéticaRESUMO
Purified Escherichia coli agmatinase (EC 3.5.3.11) expressed the same activity in the absence or presence of added Mn2+ (0-5mM). However, it was strongly inhibited by Co2+, Ni2+, and Zn2+ and almost half inactivated by EDTA. Partial inactivation by EDTA yielded enzyme species containing 0.85 +/- 0.1 Mn2+/subunit, and it was accompanied by a decrease in intensity of fluorescence emission and a red shift from the emission maximum of 340 nm to 346 nm, indicating the movement of tryptophane residues to a more polar environment. The activity and fluorescence properties of fully activated agmatinase were restored by incubation of dialysed species with Mn2+. Manganese-free species, obtained by treatment with EDTA and guanidinium chloride (3 M), were active only in the presence of added Mn2+. Results obtained, which represent the first demonstration of the essentiality of Mn2+ for agmatinase activity, are discussed in connection with a possible binuclear metal center in the enzyme.
Assuntos
Escherichia coli/enzimologia , Manganês/metabolismo , Ureo-Hidrolases/metabolismo , Catálise , Proteínas Recombinantes/metabolismo , Espectrometria de FluorescênciaRESUMO
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.