Insights on the participation of Glu256 and Asp204 in the oligomeric structure and cooperative effects of human arginase type I.

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and requires a bivalent cation, especially Mn2+ for its catalytic activity. It is a component of the urea cycle and regulates the intracellular levels of l-arginine, which makes the arginase a target for treatment of vascular diseases and asthma. Mammalian arginases contain an unusual S-shaped motif located at the intermonomeric interface. Until now, the studies were limited to structural role of the motif. Then, our interest was focused on functional aspects and our hypothesis has been that the motif is essential for maintain the oligomeric state, having Arg308 as a central axis. Previously, we have shown that the R308A mutant is monomeric and re-associates to the trimeric-cooperative state in the presence of low concentrations of guanidine chloride. We have now mutated Asp204 that interacts with Arg308 in the neighbor subunit, and also we mutated Glu256, proposed as important for oligomerization. Concretely, the human arginase I mutants D204A, D204E, E256A, E256Q and E256D were generated and examined. No differences were observed in the kinetic parameters at pH 9.5 or in tryptophan fluorescence. However, the D204A and E256Q variants were monomeric. On the other hand, D204E and E256D proved to be trimeric and kinetically cooperative at pH 7.5, whereas hyperbolic kinetics was exhibited by E256A, also trimeric. The results obtained strongly support the importance of the interaction between Arg255 and Glu256 in the cooperative properties of arginase, and Asp204 would be relevant to maintain the oligomeric state through salt bridges with Arg255 and Arg308.

Evidence for an inhibitory LIM domain in a rat brain agmatinase-like protein.

We recently cloned a rat brain agmatinase-like protein (ALP) whose amino acid sequence greatly differs from other agmatinases and exhibits a LIM-like domain close to its carboxyl terminus. The protein was immunohistochemically detected in the hypothalamic region and hippocampal astrocytes and neurons. We now show that truncated species, lacking the LIM-type domain, retains the dimeric structure of the wild-type protein but exhibits a 10-fold increased k(cat), a 3-fold decreased K(m) value for agmatine and altered intrinsic tryptophan fluorescent properties. As expected for a LIM protein, zinc was detected only in the wild-type ALP (∼2 Zn(2+)/monomer). Our proposal is that the LIM domain functions as an autoinhibitory entity and that inhibition is reversed by interaction of the domain with some yet undefined brain protein.

Studies on the functional significance of a C-terminal S-shaped motif in human arginase type I: essentiality for cooperative effects.

The functional significance of a C-terminal S-shaped motif (residues 304-322) in human arginase I was explored by examining the kinetic properties of the R308A mutant and truncated species terminating in either Arg-308 or Ala-308. Replacement of Arg-308 with alanine, with or without truncation, yielded monomeric species. All mutants were kinetically indistinguishable from the wild-type enzyme at the optimum pH of 9.5. At the more physiological, pH 7.5, hyperbolic kinetics was observed for all the mutants, in contrast with the cooperative behavior exhibited by the wild-type species. In the presence of 2mM guanidinium chloride (Gdn(+)), the single mutant R308A changed to a trimeric and kinetically cooperative form, whereas the other enzyme variants were not altered. The S-shaped motif is suggested as essential for the cooperative response of the enzyme to l-arginine at pH 7.5. Gdn(+) is suggested to mimic the guanidine group of Arg-308 at the monomer-monomer interface.