Template-binding site of AMV reverse transcriptase and inactivation of the enzyme by N-ethylmaleimide

$\text{N-}\text{Ethylmaleimide}$, a sulfhydryl-specific reagent, strongly inhibits AMV reverse transcriptase by specifically interfering with the template-binding site of the enzyme. However, the kinetics of inhibition differ widely with the composition and structure of the templates employed. The copying of templates with multiple 3′-hydroxyl termini appeared to be more susceptible to $\text{N-}\text{ethylmaleimide}$ treatment, suggesting that the reagent may interfere with initiation of DNA synthesis. The ability of a template bound to enzyme prior to $\text{N-}\text{ethylmaleimide}$ treatment to protect against inactivation of copying of other templates also, implies a common binding site for the different templates. Template exchange experiments demonstrated competition between activated calf thymus DNA and rA_n · dT_12–18 for binding to the enzyme. Thus, templates varying widely in composition and conformation appear to bind at a common site on reverse transcriptase. The experimental data also show suggestive evidence for small but finite differences in the requirements for optimal binding for templates of different structures.