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Functional consequences of a gene duplication and fusion event in an arginine kinase

Deanne M. Compaan^* and W. Ross Ellington

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

View larger version (58K): [in a new window]   Fig. 1. Multiple sequence alignment of Ensis directus arginine kinase (AK) domains 1 and 2 (D1 and D2, respectively), Limulus polyphemus AK and an AK consensus sequence (Ellington and Bush, 2002). Numbering is according to that of L. polyphemus AK. The 18 sequences used for the AK consensus are sea anemone Anthopleura domains 1 and 2 (O015992), crab Eriocheir (AAF43438), crab Carcinus (AAD48470), lobster Homarus (P14208), shrimp Penaeus (P51545), locust Schistocerca (P91798), bee Apis (PC6506), horseshoe crab Limulus (S52098), fruit fly Drosophila (P48610), trypanosome Trypanosoma (AAF23164), snail Turbo (O015989), abalone Suluculus (S46407), snail Cellana (BAB41096), sea hare Aplysia (BAB41095), squid Seipiateuthis (BAA95610), octopus Octopus (BAA95609) and chiton Lilophura (O15990). Catalytically important residues are shown in red. These residues contact and stabilize the reactants during catalysis. With the exception of Gly64, all the catalytically important residues are conserved in both domains of the Ensis contiguous dimer, suggesting two active domains. Residues shown in pink are those that interact with one another by forming salt bridges when the enzyme is in the closed state. The `reactive' cysteine characteristic of all phosphagen kinases is shown in green.

View larger version (28K): [in a new window]   Fig. 2. Summary of the nature and outcome of the expression of Ensis full-length arginine kinase (AK), truncated D1 and D2 and the various AK NusA–His-tag fusion constructs and enterokinase cleavage products.

View larger version (21K): [in a new window]   Fig. 3. Primary kinetic plots from Ensis directus full-length arginine kinase (AK). At each of six different fixed concentrations of substrate A, substrate B is varied through six concentrations. The inverse of reaction velocities (V) from this 6x6 matrix are plotted versus the inverse substrate concentration (ADP or arginine phosphate). Slopes and y-intercepts from each of the six lines in the primary plot are replotted as secondary plots to yield the kinetic constants (Ka and Kia) for each substrate as well as the Vmax of the enzyme. Note that the six lines in the primary plots intersect at or below the origin indicating no, or even negative, synergy of substrate binding.

View larger version (25K): [in a new window]   Fig. 4. Primary kinetic plots from Limulus polyphemus arginine kinase (AK) WTrev. This AK is a typical 40-kDa monomer. Its kinetic parameters were determined to serve as a source of comparison for Ensis AK. Limulus AK displays significant synergy of substrate binding, which is characteristic of both arginine kinase and creatine kinase. The intersection of the six lines in each plot above the x-axis is indicative of this substrate binding synergy. To determine kinetic constants, the slopes and y-intercepts from these primary plots were replotted in secondary plots.