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Cytoplasmic 5'-nucleotidase catalyzes acyclovir phosphorylation.

P M Keller, S A McKee, J A Fyfe

    The Journal of Biological Chemistry
    |July 25, 1985
    PubMed
    Summary

    A cytoplasmic 5'-nucleotidase enzyme phosphorylates acyclovir (ACV), an antiherpes drug, at low levels. This activity, though inefficient, explains ACV phosphate formation in uninfected cells, suggesting a potential metabolic pathway.

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    Area of Science:

    • Biochemistry
    • Enzymology
    • Pharmacology

    Background:

    • Cytoplasmic 5 '-nucleotidases (EC 3.1.3.5) are known to phosphorylate inosine.
    • Acyclovir (ACV) is a nucleoside analog with significant antiherpes activity.
    • The metabolic fate of ACV in uninfected cells, particularly its phosphorylation, is not fully understood.

    Purpose of the Study:

    • To investigate whether purified cytoplasmic 5 '-nucleotidase can catalyze the phosphorylation of acyclovir (ACV).
    • To characterize the kinetic properties of ACV phosphorylation by this enzyme.
    • To determine if this enzymatic activity can account for trace levels of ACV phosphates in cells.

    Main Methods:

    • Purification of cytoplasmic 5 '-nucleotidase from rat liver.
    • Enzymatic assays to measure ACV phosphorylation.
    • Co-chromatography, size exclusion chromatography, and gel electrophoresis to assess enzyme purity and activity.
    • Kinetic analysis including pH optimum, substrate inhibition, and competitive inhibition studies.

    Main Results:

    • ACV phosphorylating activity co-purified with cytoplasmic 5 '-nucleotidase.
    • The enzyme exhibited similar pH optimum, ATP stimulation, and phosphate inhibition for both inosine and ACV.
    • ACV phosphorylation was competitively inhibited by inosine, indicating a common catalytic site.
    • While ACV phosphorylation was inefficient (low Vmax, high Km), it was sufficient to explain observed ACV phosphate levels in cells.

    Conclusions:

    • Cytoplasmic 5 '-nucleotidase possesses the ability to phosphorylate acyclovir (ACV).
    • The kinetic parameters suggest an inefficient but potentially significant role in cellular ACV metabolism.
    • This enzymatic activity may contribute to the formation of ACV phosphates in uninfected cells.

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