FUNCTIONAL ASSIGNMENT BY CHIMERA CONSTRUCTION OF THE DOMAIN AFFECTINGHETEROTROPIC ACTIVATION OF DEOXYADENOSINE KINASE FROM LACTOBACILLUS-ACIDOPHILUS R-26

The heterodimeric subunits of deoxyadenosine kinase (dAK)-deoxyguanosi ne kinase (dGrK) from Lactobacillus acidophilus R-26 exhibit contrasti ng 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 at. (1997) J. Biol. Chem. 272, 6890-6897]. In an attempt to ide ntify domains differentiating the specificities of dAK and dGK, we con structed several chimeras splicing heterodimeric dAK within this regio n. In Chimera-III, dAK residues 120-170 were replaced by the homologou s section of dGK. dAK activity was elevated 40%, but although it retai ned its original specificity and K-m values, it could no longer be act ivated by deoxyguanosine. Moreover, both the activated dAK; and the '' dAK'' of Chimera-ID exhibited (i) an increased K-s for the leading sub strate ATP-Mg2+, suggesting the formation of intermediate enzyme speci es along their respective kinetic pathways, and (ii) broadened and low er pH optima for the dAK activities, These observations further indica te the importance of dAK residues 120-170, including the Ras-like segm ent, in catalysis and heterotropic activation. The other conformationa l properties of dAK (e.g. self-inactivity and MgATP being the leading substrate) were unaltered by this substitution, thus localizing the re sponsible domains even further upstream.