Active site studies of human immunodeficiency virus reverse transcriptase

Laura Louise Watts Mitchell, University of Pennsylvania


The active site structure of the Human Immunodeficiency Virus Reverse Transcriptase (HIV RT) was probed using chemical modification. HIV RT has two activities, a polymerase activity and an RNAse H activity. The possibility of differentiating and localizing these sites was explored using three group-specific reagents: pyridoxal 5$\sp\prime$ phosphate (PLP), which reacts with lysine residues, phenylglyoxal (PG), which reacts with arginines, and Nethylmaleimide (NEM), which reacts with cysteines. In connection with the study of PLP inhibition of the enzyme, initial rate kinetics were also performed to further characterize the system. The conclusions of this work on the polymerase activity are as follows: (1) HIV RT follows an ordered bi-bi rate equation; (2) PLP is a noncompetitive inhibitor, contrary to published work (Basu et al., 1989); (3) The inactivation of HIV RT by PG can be completely protected against by adding primer-template, and is thus indicative of an active site arginine, which was localized to position 277 in the primary structure; and (4) NEM shows a differential effect dependent on the HIV RT isolate; examination of the primary structure indicates a non-conserved cysteine at position 162 to be implicated in the active site structure, though it is not essential for activity. None of the reagents studied above had any affect on the RNAse H activity. Therefore the two activities do not share an active site, as can be differentiated between their differing sensitivities to the chemical modification reagents PLP, PG and NEM.

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Recommended Citation

Mitchell, Laura Louise Watts, "Active site studies of human immunodeficiency virus reverse transcriptase" (1991). Dissertations available from ProQuest. AAI9125721.