ABSTRACT

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

FOOTNOTES

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This work was supported in part by National Institutes of Health Grants CA-47828 and GM49635. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: Dept. of Biochemistry, State University of New York at Buffalo, Buffalo, NY 14214.

¶

Present address: Dept. of Biochemistry, Molecular and Cellular Biology, Northwestern University, Evanston, IL 60208-3520.

**

To whom correspondence should be addressed: Dept. of Biochemistry, The Ohio State University, 484 W. 12th Ave., Columbus, OH 43210-1292. Tel.: 614-292-0485; Fax: 614-292-6773.

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The abbreviations used are: TK, thymidine kinase; dGK, deoxyguanosine kinase; dAK, deoxyadenosine kinase; dCK, deoxycytidine kinase; UMCE, unmodified cloned enzyme; HSV, herpes simplex virus.

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S. Ikeda, personal communication.

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