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 deoxygua nosine kinase/deoxyadenosine kinase (dGK/dAK) of Lactobacillus acidoph ilus R-26 has begun with the Asp-Arg-Ser (DRS) motif. Replacement of A sp-78 of both subunits with Glu, Ala, or Asn reduced dGK and dAK activ ities to less than 0.2%, whereas replacement of Arg-79 with Lys, eithe r on both subunits in tandem (R79K), or on the dGK subunit only (R79K: dGK), yielded active but kinetically modified enzymes. These were part ially purified, and their kinetic and regulatory properties were analy zed, 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 slig htly reduced K-m for MgATP, The V/K efficiency ratio of dAK was also i ncreased 29-fold, but that of dGK was decreased to 5-10% due to a 10-f old 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 t hat of dAdo, but dGK modification appears to produce a stimulatory con formational effect on the opposite subunit, resembling the known unidi rectional activation of dAK by either dGuo or dGTP.