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 V-max of the R79K:dGK enzyme was increased 28-fold, with no change in the limiting K-m, for dAdo, but with a slightly reduced K-m 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 K-m, for dGuo and a reduced V-max. 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.