The mechanism of inhibition of avian myeloblastosis virus reverse transcriptase by a dialdehyde derivative of ATP. Inactivation of essential sulfhydryl group function.

Abstract:

The dialdehyde derivative of ATP inhibits DNA synthesis by AMV reverse transcriptase, while the polymerase-associated ribonuclease H activity is significantly resistant to this reagent. Neither ATP nor its dialcohol form effectively block DNA synthesis, indicating that the aldehyde moiety is required for inhibition. The nature of the reactivity of dialdehyde-ATP with AMV reverse transcriptase has been examined and we find that: (a) inhibition is non-competitive with respect to substrate deoxynucleoside triphosphate concentration, suggesting that dialdehyde-ATP does not react at the substrate binding site; (b) pretreatment of enzyme with dialdehyde-ATP or sulfhydryl group binding reagents results in the complete loss of its template binding activity; however, treatment of preformed enzyme-template-primer complex with both inhibitors did not dissociate this complex; (c) the inhibitory effect of dialdehyde-ATP was completely reversed upon addition of reducing agents, such as dithiothreitol and sodium borohydride, indicating that dialdehyde-ATP reacts with the sulfhydryl groups present in AMV reverse transcriptase; (d) comparative studies carried out with the classical sulfhydryl reagent, dithiobisnitrobenzoic acid, revealed a remarkable similarity in its action to that of dialdehyde-ATP. We therefore conclude that the dialdehyde-ATP-mediated inhibition of AMV DNA polymerase is effected via blockage of essential sulfhydryl groups present in the enzyme protein.

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