Directed mutagenesis of deoxyguanosine site at arginine 79 up-regulates turnover on deoxyadenosine kinase subunit of heterodimeric enzyme from Lactobacillus acidophilus R26.
Examination of conserved motifs on the cloned subunits of the deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) of Lactobacillus acidophilus R-26 has begun with the Asp-Arg-Ser (DRS) motif. Replacement of Asp-78 of both subunits with Glu, Ala, or Asn reduced dGK and dAK activities to less than 0.2%, whereas replacement of Arg-79 with Lys, either on both subunits in tandem (R79K), or on the dGK subunit only (R79K:dGK), yielded active but kinetically modified enzymes. These were partially purified, and their kinetic and regulatory properties were analyzed. For dAK activity, the Vmax of the R79K:dGK enzyme was increased 28-fold, with no change in the limiting Km for dAdo, but with a slightly reduced Km for MgATP. The V/K efficiency ratio of dAK was also increased 29-fold, but that of dGK was decreased to 5-10% due to a 10-fold increase in Km for dGuo and a reduced Vmax. Therefore, the R79K substitution seems to have a greater effect on dGuo binding than on that of dAdo, but dGK modification appears to produce a stimulatory conformational effect on the opposite subunit, resembling the known unidirectional activation of dAK by either dGuo or dGTP.
Hong
Y S
YS
Department of Biochemistry, Ohio State University, Columbus 43210-1292.
Ma
G T
GT
Ives
D H
DH
D001483:Base Sequence; D001665:Binding Sites; D003849:Deoxyguanosine / Q000378:metabolism*; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D015854:Up-Regulation
D017853:Phosphotransferases (Alcohol Group Acceptor); C019165:deoxyguanosine kinase; C020005:deoxyadenosine kinase; D003849:Deoxyguanosine