Tài liệu Báo cáo khoa học: Insights into the interaction of human arginase II with substrate and

Insights into the interaction of human arginase II with

substrate and manganese ions by site-directed

mutagenesis and kinetic studies

Alteration of substrate specificity by replacement of Asn149

with Asp

Vasthi Lo´ pez, Ricardo Alarco´ n, Marı´a S. Orellana, Paula Enrı´quez, Elena Uribe, Jose´ Martı´nez and

Nelson Carvajal

Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Ciencias Biolo´gicas, Universidad de Concepcio´n, Chile

Arginase (l-arginine urea amidino hydrolase,

EC 3.5.3.1) catalyzes the hydrolysis of l-arginine to

yield l-ornithine and urea, and exhibits an absolute

requirement for bivalent metal ions, especially Mn2+,

for catalytic activity. Metal ions are thought to acti￾vate a coordinated water molecule, by lowering the

pKa for proton ionization and generation of the

hydroxide that nucleophilically attacks the guanidino

carbon of the scissile bond of l-arginine [1–3].

The enzyme is widely distributed in living organisms,

where it serves several functions, including ureagenesis

and regulation of the cellular levels of l-arginine, a

precursor for the production of creatine, proline, poly￾amines and nitric oxide [4–7]. Mammalian tissues con￾tain two distinct isoenzymic forms: arginase I, which is

highly expressed in the liver and it has been tradition￾ally associated with ureagenesis, and the extrahepatic

arginase II, which is thought to provide a supply of

l-ornithine for proline and polyamine biosynthesis

[7–12]. Both arginase isoforms are also thought to par￾ticipate in the regulation of nitric oxide biosynthesis by

competing with nitric oxide synthases for the common

Keywords

manganese ions; histidine; agmatinase

activity; arginase II; human

Correspondence

N. Carvajal, Departamento de Bioquı´mica y

Biologı´a Molecular, Facultad de Ciencias

Biolo´gicas, Universidad de Concepcio´n,

Casilla 160-C, Concepcio´n, Chile

Fax: +56 41 239687

E-mail: [email protected]

(Received 24 May 2005, revised 13 July

2005, accepted 19 July 2005)

doi:10.1111/j.1742-4658.2005.04874.x

To examine the interaction of human arginase II (EC 3.5.3.1) with sub￾strate and manganese ions, the His120Asn, His145Asn and Asn149Asp

mutations were introduced separately. About 53% and 95% of wild-type

arginase activity were expressed by fully manganese activated species of the

His120Asn and His145Asn variants, respectively. The Km for arginine (1.4–

1.6 mm) was not altered and the wild-type and mutant enzymes were essen￾tially inactive on agmatine. In contrast, the Asn149Asp mutant expressed

almost undetectable activity on arginine, but significant activity on agma￾tine. The agmatinase activity of Asn149Asp (Km ¼ 2.5 ± 0.2 mm) was

markedly resistant to inhibition by arginine. After dialysis against EDTA,

the His120Asn variant was totally inactive in the absence of added Mn2+

and contained < 0.1 Mn2+Æsubunit)1

, whereas wild-type and His145Asn

enzymes were half active and contained 1.1 ± 0.1 Mn2+Æsubunit)1 and

1.3 ± 0.1 Mn2+Æsubunit)1

, respectively. Manganese reactivation of metal￾free to half active species followed hyperbolic kinetics with Kd of

1.8 ± 0.2 · 10)8 m for the wild-type and His145Asn enzymes and

16.2 ± 0.5 · 10)8 m for the His120Asn variant. Upon mutation, the chro￾matographic behavior, tryptophan fluorescence properties (kmax ¼ 338–

339 nm) and sensitivity to thermal inactivation were not altered. The

Asn149fiAsp mutation is proposed to generate a conformational change

responsible for the altered substrate specificity of arginase II. We also con￾clude that, in contrast with arginase I, Mn2+A is the more tightly bound

metal ion in arginase II.

4540 FEBS Journal 272 (2005) 4540–4548 ª 2005 FEBS