DNA polymerase activity of hepatitis B virus particles: differential inhibition by L-enantiomers of nucleotide analogs.

DNA polymerase activity was assayed in hepatitis B virus (HBV) and core particles isolated from chronic producer lines. The particle-associated DNA polymerase activity, which was found to be limited to incorporation of only a few nucleotides, was inhibited by the 5'-triphosphates of nucleoside analogs. The 1-beta-L (1S,4R) and 1-beta-D (1R,4S) enantiomers of antiviral nucleoside analogs were compared for the ability to inhibit incorporation of natural nucleoside triphosphates into the viral DNA. Previously, both enantiomers of several analogs were found to be substrates for human immunodeficiency virus type 1 reverse transcriptase (HIV RT); the 1-beta-D enantiomers of some pairs were preferred as substrates. In contrast, the 1-beta-L enantiomers of all pairs tested were the more potent inhibitors of labeled substrate incorporation into hepatitis B virus DNA; the concentration required to inhibit the incorporation of the natural substrate by 50% was 6-fold to several hundred-fold lower than the concentration of the 1-beta-D enantiomer required for the same inhibitory effect. This preference for the 1-beta-L enantiomers was observed for both RNA-directed synthesis in core particles and DNA-directed synthesis in viral particles. The observed antiviral effect of the nucleoside analogs in cell culture seemed to be limited chiefly by their phosphorylation in cells.

Beta-L-thymidine 5'-triphosphate analogs as DNA polymerase substrates.

beta-L-3'-Deoxythymidine 5'-triphosphate (L-ddTTP) and beta-L-3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (L-d4TTP) were substrates for human immunodeficiency virus reverse transcriptase, Escherichia coli DNA polymerase I (Klenow), and Sequenase (modified T7 DNA polymerase). The beta-D- and beta-L-enantiomers of 5-methyluridine 5'-triphosphate (rTTP) were inhibitors but not substrates of reverse transcriptase. The steady-state Km values for L-ddTTP and L-d4TTP, with all three enzymes, were 12-70-fold larger than the Km values for the corresponding D-enantiomers. The Km value of reverse transcriptase for L-ddTTP was 50-fold larger than that for D-ddTTP because the Kd for L-ddTTP was 5-fold larger than that for D-ddTTP, and the first-order rate constant for incorporation of L-ddTMP into the template-primer was 10% that of the D-enantiomer. The D- and L-enantiomers had kcat values with reverse transcriptase and Sequenase that were similar to kcat for the natural substrate, thymidine 5'-triphosphate (dTTP). Thus, the rate determining step appeared to be dissociation of the enzyme-chain-terminated template-primer complex. In contrast, kcat values for the L-enantiomers with Klenow were only 0.1% that of dTTP, and the kcat values for the D-enantiomers were 15% the kcat for dTTP. The reduced kcat values were due to a change in rate determining step from dissociation of the Klenow-chain-terminated template-primer complex to an earlier step in the reaction mechanism, presumably catalysis. Thus, these DNA polymerases did not stereospecifically recognize D-nucleoside 5'-triphosphate analogs as substrates.

Phosphorylation of carbovir enantiomers by cellular enzymes determines the stereoselectivity of antiviral activity.

Two enantiomers of carbovir, a carbocyclic analog of 2',3'-dideoxyguanosine, were compared with respect to their phosphorylation and the phosphorylation of their nucleotides by mammalian enzymes. 5'-Nucleotidase catalyzed the phosphorylation of (-)-carbovir, which is active against HIV (human immunodeficiency virus), but did not phosphorylate (+)-carbovir. (-)-Carbovir monophosphate was 7,000 times more efficient as a substrate for GMP kinase than was (+)-carbovir monophosphate. Pyruvate kinase, phosphoglycerate kinase, and creatine kinase phosphorylated both enantiomers of carbovir diphosphate at similar rates. Nucleoside-diphosphate kinase preferentially phosphorylated the (-)-enantiomer. Both enantiomers of carbovir triphosphate were substrates and alternative substrate inhibitors of HIV reverse transcriptase. Thus, the contrasting HIV-inhibitory activities of carbovir enantiomers were due to differential phosphorylation by cellular enzymes and not due to enantioselectivity of HIV reverse transcriptase.

Reduced S-adenosylhomocysteine hydrolase. Kinetics and thermodynamics for binding of 3'-ketoadenosine, adenosine, and adenine.

S-Adenosylhomocysteine hydrolase (SAHase) was resolved into apoenzyme and NAD+ by acidic ammonium sulfate treatment. The apoenzyme was catalytically inactive, but could be reconstituted to active enzyme with NAD+. Reduced SAHase (ENADH) that was prepared by reconstitution of the apoenzyme with NADH was catalytically inactive. ENADH was oxidized by 3'-ketoadenosine to active SAHase. The recovery of activity paralleled the oxidation of enzyme-bound NADH. The association rate constant for ENADH and 3'-ketoadenosine was 6.1 x 10(2) M-1 s-1, and the dissociation rate constant was calculated to be 4 x 10(-7) s-1. This association rate constant was considerably smaller than the association rate constant for adenosine and SAHase (greater than 10(7) M-1 s-1). However, the observed pseudo first-order rate constant for reaction of 3'-ketoadenosine with ENADH (0.6 s-1 with 1 mM 3'-ketoadenosine) approached kcat for the hydrolytic reaction (1.2 s-1). Thus, bound 3'-ketoadenosine probably reacted sufficiently rapidly with ENADH to be considered a kinetically competent intermediate. The dissociation constants of SAHase for adenosine and 4',5'-dehydroadenosine, substrates for the enzyme, were 9 and 14 microM, respectively. In contrast, the dissociation constants of ENADH for 3'-ketoadenosine and 4',5'-dehydro-3'-ketoadenosine, intermediates of the catalytic reaction, were significantly lower with values of 600 and 300 pM, respectively. The equilibrium constant for reduction of enzyme-bound NAD+ in the absence of an adenosine analogue, as estimated from cyanide binding studies, was 10-fold more favorable than that for free NAD+. ENADH was highly fluorescent (emission maximum 428 nm, excitation 340 nm) with a quantum yield that was six times that of free NADH. Since SAHase reduced by adenosine was not highly fluorescent, enzyme-bound intermediates quenched the fluorescence of enzyme-bound NADH. Adenosine and adenine quenched the fluorescence of ENADH. Cyanide formed a complex with SAHase that was analogous to ENADH. Adenine stabilized this complex sufficiently that addition of 65 microM adenine and 25 mM cyanide to SAHase caused total complex formation with loss of over 95% of the catalytic activity.

Mechanism of bovine liver S-adenosylhomocysteine hydrolase. Steady-state and pre-steady-state kinetic analysis.

The kinetic mechanism of S-adenosylhomocysteine hydrolase was investigated by stopped-flow spectrofluorometry at pH 7.0 and 25 degrees C. Pre-steady-state kinetic steps were identified with chemical steps proposed for the mechanism of this enzyme (Palmer, J.L., and Abeles, R.H. (1979) J. Biol. Chem. 254, 1217-1226). The steady-state kinetic constants for the hydrolysis or synthesis of S-adenosylhomocysteine were in good agreement with those values calculated from the pre-steady-state rate constants. The equilibrium constant for dehydration of 3'-ketoadenosine to 3'-keto-4',5'-dehydroadenosine on the enzyme was 3. The analogous equilibrium constant for addition of L-homocysteine to S-3'-keto-4',5'-dehydroadenosylhomocysteine on the enzyme was 0.3. The elimination of H2O from adenosine in solution had an equilibrium constant of 1.4 (aH2O = 1). Thus, the equilibrium constants for these elimination reactions on the enzyme were probably not perturbed significantly from those in solution. The equilibrium constant for the reduction of enzyme-bound NAD+ by adenosine was 8, and the analogous constant for the reduction of the enzyme by S-adenosylhomocysteine was 4. The equilibrium constant for the reduction of NAD+ by a secondary alcohol in solution was 5 x 10(-5) at pH 7.0. Consequently, the reduction of enzyme-bound NAD+ by adenosine was 10(5)-fold more favorable than the reduction of free NAD+. The magnitude of the first-order rate constants for the interconversion of enzyme-bound intermediates varied over a relatively small range (3-80 s-1). Similarly, the magnitude of the equilibrium constants among enzyme-bound intermediates varied over a narrow range (0.3-10). These results were consistent with the overall reversibility of the reaction.

Characterization of a unique tomaymycin-d(CICGAATTCICG)2 adduct containing two drug molecules per duplex by NMR, fluorescence, and molecular modeling studies.

Tomaymycin is a member of the pyrrolo[1,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. This antibiotic is proposed to react with the exocyclic 2-amino group (N2) of guanine to form a covalent adduct that lies snugly within the minor groove of DNA. While DNA-footprinting experiments using methidiumpropyl-EDTA have revealed the favored bonding sequences for tomaymycin and related drugs on DNA, the stereochemistry at the covalent bonding site (C-11) and orientation in the minor groove were not established by these experiments. In previous studies using a combined fluorescence, high-field NMR, and molecular modeling approach, we have shown that for tomaymycin there are two diastereomeric species (11R and 11S) on both calf thymus DNA and d(ATGCAT)2. Although we were able to infer the identity (stereochemistry at C-11 and orientation in the minor groove) of the two species on d(ATGCAT)2 by high-field NMR and fluorescence studies, in combination with molecular mechanics calculations, definitive experimental evidence was lacking. We have designed and synthesized a self-complementary 12-mer [d(CICGAATTCICG)2] based on the Dickerson dodecamer [d(CGCGAATTCGCG)2] that bonds identically two tomaymycin molecules, each having a defined orientation and stereochemistry. Thus the bis(tomaymycin)-12-mer adduct maintains the self-complementarity of the original duplex molecule. Two-dimensional proton J-correlated spectroscopy (COSY) of the bis(tomaymycin)-d(CICGAATTCICG)2 adduct (I = inosine) unequivocally shows that C-11 of tomaymycin covalently bonds through N2 of guanine with an 11S stereochemistry in the sequence 5'-CGA-3'.(ABSTRACT TRUNCATED AT 250 WORDS)