Functional assignment by Chimera construction of the domain affecting heterotropic activation of deoxyadenosine kinase from Lactobacillus acidophilus R-26.
The heterodimeric subunits of deoxyadenosine kinase (dAK)-deoxyguanosine kinase (dGK) from Lactobacillus acidophilus R-26 exhibit contrasting conformations manifested in the nearly unidirectional heterotropic activation of dAK when dGK binds deoxyguanosine. This is mediated, in part, by the conserved Ras switch I-like sequence (residues 153-161) [Guo et al. (1997) J. Biol. Chem. 272, 6890-6897]. In an attempt to identify domains differentiating the specificities of dAK and dGK, we constructed several chimeras splicing heterodimeric dAK within this region. In Chimera-III, dAK residues 120-170 were replaced by the homologous section of dGK. dAK activity was elevated 40%, but although it retained its original specificity and Km values, it could no longer be activated by deoxyguanosine. Moreover, both the activated dAK and the "dAK" of Chimera-III exhibited (i) an increased Ks for the leading substrate ATP-Mg2+, suggesting the formation of intermediate enzyme species along their respective kinetic pathways, and (ii) broadened and lower pH optima for the dAK activities. These observations further indicate the importance of dAK residues 120-170, including the Ras-like segment, in catalysis and heterotropic activation. The other conformational properties of dAK (e.g. self-inactivity and MgATP being the leading substrate) were unaltered by this substitution, thus localizing the responsible domains even further upstream.
Guo
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Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210-1292, USA.
Ives
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DH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007779:Lactobacillus acidophilus / Q000201:enzymology*; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020005:deoxyadenosine kinase