A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase

Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3′-azido-2′,3′-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5′-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s−1) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 µM) and chemistry forward and reverse reactions, 0.38 and 0.22 s−1, respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/Km (7 × 10−6 μ M−1 s−1). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5′-triphosphate (8-oxo-dGTP) show this same phenomena

Exonuclease Removal of Dideoxycytidine (Zalcitabine) by the Human Mitochondrial DNA Polymerase▿

The toxicity of nucleoside analogs used for the treatment of human immunodeficiency virus infection is due primarily to the inhibition of replication of the mitochondrial genome by the human mitochondrial DNA polymerase (Pol γ). The severity of clinically observed toxicity correlates with the kinetics of incorporation versus excision of each analog as quantified by a toxicity index, spanning over six orders of magnitude. Here we show that the rate of excision of dideoxycytidine (zalcitabine; ddC) was reduced fourfold (giving a half-life of ∼2.4 h) by the addition of a physiological concentration of deoxynucleoside triphosphates (dNTPs) due to the formation of a tight ternary enzyme-DNA-dNTP complex at the polymerase site. In addition, we provide a more accurate measurement of the rate of excision and show that the low rate of removal of ddCMP results from both the unfavorable transfer of the primer strand from the polymerase to the exonuclease site and the inefficient binding and/or hydrolysis at the exonuclease site. The analogs ddC, stavudine, and ddATP (a metabolite of didanosine) each bind more tightly at the polymerase site during incorporation than normal nucleotides, and this tight binding contributes to slower excision by the proofreading exonuclease, leading to increased toxicity toward mitochondrial DNA

A R T I C L E S

The predominant biosynthetic route to vitamin B6 is catalyzed by a single enzyme. The synthase subunit of this enzyme, Pdx1, operates in concert with the glutaminase subunit, Pdx2, to catalyze the complex condensation of ribose 5-phosphate, glutamine and glyceraldehyde 3-phosphate to form pyridoxal 5¢-phosphate, the active form of vitamin B6. In previous studies it became clear that many if not all of the reaction intermediates were covalently bound to the synthase subunit, thus making them difficult to isolate and characterize. Here we show that it is possible to follow a single turnover reaction by heteronuclear NMR using 13 C-and 15 N-labeled substrates as well as 15 N-labeled synthase. By denaturing the enzyme at points along the reaction coordinate, we solved the structures of three covalently bound intermediates. This analysis revealed a new 1,5 migration of the lysine amine linking the intermediate to the enzyme during the conversion of ribose 5-phosphate to pyridoxal 5¢-phosphate. Vitamin B6 is a composite term for six different vitamers recognized as pyridoxine (1), pyridoxal (2), pyridoxamine (3) and their corresponding 5¢-phosphorylated derivatives (4, 5 and 6, respectively). The vitamer pyridoxal 5¢-phosphate (PLP; 5) is known for its catalytic versatility 1 . In most cases PLP acts as an enzyme-bound cofactor that participates in diverse biochemical reactions and pathways, including amino acid biosynthesis, carbohydrate metabolism and the modification of many amine-containing compounds. It has been estimated that at least 140 different PLP-dependent enzymes exist, and approximately 1.5% of the genes in a typical prokaryote encode PLP-using enzymes 2 . A number of these enzymes are already targets for therapeutic agents, and many more are thought to be good candidates 3 . In addition to these well-documented roles in enzyme catalysis, PLP has recently been implicated in singlet oxygen resistance As vitamin B6 is required for various processes in all organisms, it is either biosynthesized de novo, as is the case for most microorganisms and plants, or acquired externally, as is necessary for animals 6 . Two independent de novo pathways for the biosynthesis of PLP are currently known. The best understood pathway, found in Escherichia coli (1-deoxyxylulose 5-phosphate (7)-dependent), is rarely used compared with the route present in most other species (ribose 5-phosphate-dependent) 7 . Pdx1-Pdx2, the biosynthetic enzyme found in Bacillus subtilis, catalyzes the condensation reaction shown in Scheme 1 using either D-ribose 5-phosphate (R5P; 8) or D-ribulose 5-phosphate (Ru5P; 9), glutamine (10) and D-glyceraldehyde 3-phosphate (G3P; 11) Previous high-resolution mass spectrometric analysis of Pdx1 revealed that the first substrate used, R5P or Ru5P, becomes covalently attached to the enzyme through an active site lysine concomitant with the loss of water (Pdx1 + 212 Da) The first hint that the Pdx1-Z 1 intermediate was not bound via an imine was provided by the observation of its relatively high stability. It was shown that ESI-FTMS spectra of Pdx1-Z 1 could be collected even without NaBH 4 reduction 23 . This was surprising because the preparation of the sample for analysis included a reversed-phase desalting step that was performed under mildly acidic conditions that should result in imine hydrolysis 23 . Furthermore, the addition of only NH 4 Cl (or glutamine if Pdx2 was present) to Pdx1-Z 1 resulted in the accumulation of the chromophoric species, I 320 , which appeared to be bound to the enzyme via C5 (ref. 22). Based on the fact that only one molecule of water is lost during the reaction of Pdx1 with R5P to form Pdx1-Z 1 , it was difficult to imagine that the substrate was initially covalently bound to C5. The data suggested the possibility of a C-N bond shift in going from Pdx1-Z 1 to I 320 , but there was no direct structural data supporting this claim. In addition to these two intermediates, a thir

Catalysis of a new ribose carbon-insertion reaction by the molybdenum cofactor biosynthetic enzyme MoaA

MoaA, a radical S-adenosylmethionine enzyme, catalyzes the first step in molybdopterin biosynthesis. This reaction involves a complex rearrangement in which C8 of guanosine triphosphate is inserted between C2′ and C3′ of the ribose. This study identifies the site of initial hydrogen atom abstraction by the adenosyl radical and advances a mechanistic proposal for this unprecedented reaction. © 2013 American Chemical Society

Catalysis of a New Ribose Carbon-Insertion Reaction by the Molybdenum Cofactor Biosynthetic Enzyme MoaA

MoaA, a radical <i>S</i>-adenosylmethionine enzyme, catalyzes the first step in molybdopterin biosynthesis. This reaction involves a complex rearrangement in which C8 of guanosine triphosphate is inserted between C2′ and C3′ of the ribose. This study identifies the site of initial hydrogen atom abstraction by the adenosyl radical and advances a mechanistic proposal for this unprecedented reaction