The crystal structure of Torpedo californica (Tc) acetylcholinesterase (AChE) carbamoylated by the physostigmine analogue 8-(cis-2,6-dimethylmorpholino)octylcarbamoyleseroline ( MF268) is reported at 2.7 Å resolution. In the X-ray structure, the dimethylmorpholinooctylcarbamic moiety of MF268 is covalently bound to the catalytic serine, which is located at the bottom of a long and narrow gorge. The alkyl chain of the inhibitor fills the upper part of the gorge, blocking the entrance of the active site. This prevents eseroline, the leaving group of the carbamoylation process, from exiting through this path. Surprisingly, the relatively bulky eseroline is not found in the crystal structure, thus implying the existence of an alternative route for its clearance. This represents indirect evidence that a "back door" opening may occur and shows that the release of products via a "back door" is a likely alternative for this enzyme. However, its relevance as far as the mechanism of substrate hydrolysis is concerned needs to be established. This study suggests that the use of properly designed acylating inhibitors, which can block the entrance of catalytic sites, may be exploited as a general approach for investigating the existence of "back doors" for the clearance of products.

Reverse transcription of human immunodeficiency virus type 1 (HIV‐1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3′ terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV‐1 reverse transcriptase (RT)‐associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS‐containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA‐DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3′ terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co‐ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30‐fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA.

In this study, we have analyzed the interdependence between the polymerase and RNase H active sites of human immunodeficiency virus-1 reverse transcriptase (RT) using an in vitro system that closely mimics the initiation of (؉)-strand DNA synthesis. Time course experiments show that RT pauses after addition of the 12th DNA residue, and at this stage the RNase H activity starts to cleave the RNA primer from newly synthesized DNA. Comparison of cleavage profiles obtained with 3-and 5-end-labeled primer strands indicates that RT now translocates in the opposite direction, i.e. in the 5 direction of the RNA strand. DNA synthesis resumes again in the 3 direction, after the RNA-DNA junction was efficiently cleaved. Moreover, we further characterized complexes generated before, during, and after position ؉12, by treating these with Fe 2؉ to localize the RNase H active site on the DNA template. Initially, when RT binds the RNA/DNA substrate, oxidative strand breaks were seen at a distance of 18 base pairs upstream from the primer terminus, whereas 17 base pairs were observed at later stages when the enzyme binds more and more DNA/DNA. These data show that the initiation of (؉)-strand synthesis is accompanied by a conformational change of the polymerase-competent complex. Retroviral RTs1 are multifunctional enzymes possessing RNA-and DNA-dependent polymerase activities and a ribonuclease H (RNase H) activity that degrades the RNA strand of RNA/DNA hybrids (1, 2). Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) uses a cellular tRNA primer to initiate reverse transcription from a complementary primer-binding site (PBS) near the 5Ј-end of the viral RNA (3-6). Despite changes of binding and kinetic properties, observed concomitant with synthesis of the first DNA strand (7), i.e. (Ϫ)-strand DNA, complexes with the initially bound RNA/ RNA duplex and the newly synthesized DNA/RNA substrates share certain common features. RNase H cleavages on the RNA strand of DNA/RNA primer/template combinations occur at a constant distance of 18 bp upstream of the nascent primer terminus (8, 9). Analogously, RNase H-induced cleavages within the tRNA/RNA duplex, designated as RNase H* activity (10), were observed at the same distance from the 3Ј-end of the primer, although these cuts are restricted to stalled complexes (11). Together, these data provide strong evidence that RT binds to both RNA/RNA and DNA/RNA substrates with the same orientation, and the number of bp between the two active sites is 18 in each case.RT-DNA/DNA complexes, which are generated during (ϩ)-strand synthesis, have been relatively well characterized (12-15). The crystal structure of HIV-1 RT complexed to an 18-base primer/19-base template DNA homoduplex (12) suggests that the first 7 DNA/DNA base pairs near the polymerase active site adopt an A-type conformation, whereas the region further upstream is in the preferred B-conformation, both structurally distinct segments being separated by a kink.Little information is currently available regarding th...

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