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Functional consequences of a gene duplication and fusion event in an arginine kinase
Department of Biological Science and Institute of Molecular
Biophysics, Florida State University, Tallahassee, FL 32306-4370,
USA
* Present address: Genentech, 1 DNA Way, South San Francisco, CA 94080-4990,
USA
Author for correspondence (e-mail:
elling{at}bio.fsu.edu)
Accepted 19 February 2003
Arginine kinase (AK) from the foot of the razor clam Ensis directus consists of two full-length AK domains, denoted D1 and D2, fused in a single polypeptide chain. The full-length cDNA for Ensis AK was obtained and its deduced amino acid sequence was analyzed in the context of the X-ray crystal structure of a typical, monomeric AK. Both domains of Ensis AK contain most of the residues currently thought to be critical in catalysis, suggesting that both AK domains are catalytically competent. The full-length Ensis AK, a D2–NusA–His-tag fusion protein and a D2-truncated AK (enterokinase cleavage product of the fusion protein) were expressed in Escherichia coli and purified. All recombinant AK constructs displayed high enzyme activity. Attempts at expressing active D1 alone, D2 alone or a D1–NusA–His-tag fusion protein were unsuccessful. The catalytic properties of the active proteins were compared with the corresponding properties of recombinant AK from the horseshoe crab Limulus polyphemus, which is a typical monomeric AK. In contrast to expectations, the kinetic results strongly suggest that Ensis AK has only one active domain, namely D2. The Kcat values for all Ensis constructs were roughly twice that of typical AKs, indicating higher overall catalytic throughput at the competent active site. Furthermore, both the full-length and truncated D2 Ensis AKs showed no synergism of substrate binding unlike typical AKs. The D2–NusA–His-tag fusion construct actually displayed negative synergism of substrate binding, which means that, in effect, the first substrate bound acts as a competitive inhibitor of the second. The conservation of the structure of the apparently inactive D1 may be related to constraints imposed by structural changes that could potentially impact substrate binding in D2 and/or possibly influence the proper folding of the enzyme during synthesis. Overall, the results from the present study indicate that the AK contiguous dimer from Ensis directus functions with activity in only the second domain. Although lacking activity in D1, D2 appears to compensate by having a higher intrinsic catalytic throughput than typical 40-kDa monomeric AKs.
Key words: arginine kinase, gene duplication, Ensis directus, D1, D2, gene fusion, domain
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