Implications of the S-shaped domain in the quaternary structure of human arginase

Arginase I is a homotrimeric protein with a binuclear manganese cluster. At the C-terminus of each monomer, the polypeptide chain forms an unusual S-s haped oligomerization motif where the majority of intermonomer contacts are located [Z.F. Kanyo, L.R. Scolnick, D.E. Ash, D.W. Christianson, Nature 38 3 (1996) 554-557]. In order to study the implication of this motif in the q uaternary structure of human arginase I, we have constructed a truncated ar ginase lacking the 14 C-terminal amino acids, leaving Arg-308 as the last r esidue in the sequence. The resulting protein retains its trimeric structur e, as determined by gel filtration (molecular mass 94 kDa). The same result was obtained in the presence of high ionic strength (KCl 0.5 M). Both data indicate that neither the S-shaped motif nor Arg-308 are fundamental in ke eping the trimeric quaternary structure. Data obtained from intrinsic aniso tropy and fluorescence intensity studies allow us to predict that the dista nce between the two unique tryptophans in the sequence is 2.9 nm in the nat ive arginase and 4.1 nm for the truncated mutant. These distances allow us to assume a different conformational state in the truncated arginase withou t any change in its quaternary structure, suggesting that the carboxy-termi nal motif is not the most prominent domain implicated in the quaternary str ucture of human arginase. Collisional quenching studies reinforce this poss ibility, since using I- as quenching molecule we were able to distinguish t he two tryptophans in the truncated arginase. Moreover, kinetic studies sho w that the truncated mutant was fully active. In summary, the main conclusi on about the structure of the human arginase I, derived from our study, is that the C-terminal S-shaped motif is not basic to the maintenance of the q uaternary structure nor to the activity of the protein. (C) 2000 Elsevier S cience B.V. All rights reserved.