FUNCTIONAL ASSIGNMENT BY CHIMERA CONSTRUCTION OF THE DOMAIN AFFECTINGHETEROTROPIC ACTIVATION OF DEOXYADENOSINE KINASE FROM LACTOBACILLUS-ACIDOPHILUS R-26
Sy. Guo et Dh. Ives, FUNCTIONAL ASSIGNMENT BY CHIMERA CONSTRUCTION OF THE DOMAIN AFFECTINGHETEROTROPIC ACTIVATION OF DEOXYADENOSINE KINASE FROM LACTOBACILLUS-ACIDOPHILUS R-26, The Journal of biological chemistry, 273(41), 1998, pp. 26624-26630
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.