Acid-catalyzed hydration of reduced nicotinamide adenine dinucleotide and its analogues.

The rate of the primary acid modification reaction of 1,4-dihydronicotinamide adenine dinucleotide (NADH) and 1,4-dihydro-3-acetylpyridine adenine dinucleotide (APADH) and their analogues has been studied over a wide pH range (pH 1-7) with a variety of general acid catalysts. The rate depends on [H+] at moderate pH and becomes independent of [H+] at low pH. This behavior is attributed to substrate protonation at the carbonyl group (pK of NADH = 0.6). The reaction is general acid catalyzed; large solvent deuterium isotope effects are observed for the general acid and lyonium ion terms. Most buffers cause a linear rate increase with increasing buffer concentration, but certain buffers cause a hyperbolic rate increase. The nonlinear buffer effects are due to complexation of the buffer with the substrate, rather than to a change in rate-limiting step. The rate-limiting step is a proton transfer from the general acid species to the C5 position of the substrate. Anomerization is not a necessary first step in the case of the primary acid modification reaction of beta-NADH, in which beta to alpha anomerization takes place.     

Uptake and efflux of adenine and its derivatives in Neurospora crassa.

Conidia of wild-type Neurospora crassa, preincubated for 3 1/2 h in growth medium, showed a typical triphasic pattern of adenine uptake. The three phases consisted of a quick initial uptake, followed by a plateau phase, and then by a resume lowered uptake. A study of the relative influx and efflux of [14C] adenine showed that the plateau phase in fact is a period of transmembrane movement of adenine and adenine metabolites. The efflux during the plateau phase essentially cancelled out all the influx during the same period. The uptake curve derived after taking into account the effluxed portion of radioactivity indicated that the second phase represents a period of lowered uptake activity. The beginning of the lowered uptake activity during the second phase is correlated with the presence of a high intracellular level of ATP derived from exogenous [14C]adenine. At the end of the secod phase, the intracellular level of ATP is much smaller and the rate of adenine uptake increases again. Analysis of the acid-soluble pool after feeding [14C]adenine indicated the presence of other 14C-nucleotides, but no detectable levels of bases and nucleosides were present. However, chromatographic analysis of the medium indicated that efflux results essentially in the accumulation of bases. The significance of this finding in relation to efflux is discussed.     

The reactions of oxo-osmium ligand complexes with isopentenyl adenine and its nucleoside.

We report syntheses of oxo-osmium(VI)bis(ligand) esters of N6-(delta2-isopentenyl) adenine (6-ipAde) and its nucleoside (IPA) which result from the addition of OsO4 to the double bond of the isopentenyl group. A study of the kinetics of these reactions shows that under typical conditions the rates of reaction relative to thymidine are as follows: for OsO4-pyridine: thymidine = 1; 6-ipAde = 4600: for OsO4-2,2'-bipyridyl: thymidine = 380; 6-ipAde = 8600; IPA = 8600. We also report syntheses of osmate esters of IPA in which the osmium is bonded through the 2'-and 3'-hydroxyl groups of the ribose residue.     

Inhibitory and mutagenic effects of sodium nitroprusside and its adenine complex on Escherichia coli.

Sodium nitroprusside and its adenine complex were found to decrease the growth rate of exponentially growing Escherichia coli cultures, and the adenine complex to exert in addition a bactericidal effect. In mutation experiments the latter compound failed to induce base-pair substitutions in the E. coli strains tested but the results do not allow to exclude other mutagenic mechanisms.     

M.FokI methylates adenine in both strands of its asymmetric recognition sequence

M.FokI, a type-IIS modification enzyme from Flavobacterium okeanokoites, was purified, and its activity was characterized in vitro. The enzyme was found to be a DNA-adenine methyltransferase and to methylate both strands of the asymmetric FokI recognition sequence: (formula; see text) M.FokI does not methylate single-stranded DNA, nor does it methylate double-stranded DNA at sequences other than FokI sites.     

Free radical and ionic reaction of bisulfite with reduced nicotinamide adenine dinucleotide and its analogues.

1,4-Dihydronicotinamide adenine dinucleotide (NADH) and its analogues undergo two reactions in sulfite buffers in the pH range 5.5-7.1: (1) an oxygen-mediated free-radical chain reaction which results in the oxidation of the dihydropyridine to the pyridinium salt, and (2) an ionic reaction which results in the hydration of the 5,6 double bond of the dihydropyridine. The free-radical reaction is inhibited by superoxide dismutase (indicating the involvement of superoxide radicals) and by free-radical inhibitors. The ionic reaction is not affected by free-radical inhibitors and follows the rate law: rate = [substrate] [HSO3-] (K + SIGMAK' [HA]), where HA is a general acid of hydronium ion. The occurrence of third-order terms of the type [substrate] X [HSO3-] [HA] is consistent with the formation of a reactive bisulfite-substrate complex, which undergoes general acid catalyzed hydration.     

Unusual sensitivity of Escherichia coli to adenine or adenine plus histidine.

The W3110 strain of Escherichia coli K-12 is unusually sensitive to adenine. Inhibition of growth is relieved by a combination of thiamine and uridine (or cytidine). In the presence of histidine, inhibition is more severe and is relieved by a combination of thiamine, glycine, uridine (or cytidine), and inosine (or guanosine).     

Three-dimensional structure of human adenine phosphoribosyltransferase and its relation to DHA-urolithiasis

In mammals, adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) is present in all tissues and provides the only known mechanism for the metabolic salvage of adenine resulting from the polyamine biosynthesis pathway or from dietary sources. In humans, APRT deficiency results in serious kidney illness such as nephrolithiasis, interstitial nephritis, and chronic renal failure as a result of 2,8-dihydroxyadenine (DHA) precipitation in the renal interstitium. To address the molecular basis of DHA-urolithiasis, the recombinant human APRT was crystallized in complex with adenosine 5'-monophosphate (AMP). Refinement of X-ray diffraction data extended to 2.1 A resolution led to a final crystallographic R(factor) of 13.3% and an R(free) of 17.6%. This structure is composed of nine beta-strands and six alpha-helices, and the active site pocket opens slightly to accommodate the AMP product. The core of APRT is similar to that of other phosphoribosyltransferases (PRTases), although the adenine-binding domain is quite different. Structural comparisons between the human APRT and other "type I" PRTases of known structure revealed several important features of the biochemistry of PRTases. We propose that the residues located at positions corresponding to Leu159 and Ala131 in hAPRT are responsible for the base specificities of type I PRTases. The comparative analysis shown here also provides structural information for the mechanism by which mutations in the human APRT lead to DHA-urolithiasis.     

Hysteretic characteristic of adenine phosphoribosyltransferase.

Preassay-incubation of the highly purified human erythrocyte adenine phosphoribosyltransferase (EC 2.4.2.7) (AMP pyrophosphorylase) with one of its substrates, 5-phosphoribosyl 1-pyrophosphate (PRibPP), changes the apparent V max value of the enzyme reaction. The extent of inhibition by preassay-incubation with an inhibitor, fructose 1,6-diphosphate (FDP), or a destabilizer, hypoxanthine (Hx), is found not to be proportional to the amount of the inhibitor present. The maximum inhibition achieved by preassay-incubation was about 40%. The PRibPP, FDP, and Hx induced changes in AMP pyrophosphorylase do not require the presence of divalent ions. The inhibtion of AMP pyrophosphorylase produced by preincubation with Hx was prevented when PRibPP was added to the preassay-incubation system. However, the preassay-incubation effect of FDP was only partially diminished under the same conditions. Contrary to the PRibPP-bound AMP pyrophosphorylase, the adenine-bound enzyme was found to be more heat labile than the unbound enzyme. Similar thermal instability was also observed with FDP- and Hx-bound enzyme. Our experimental results indicate that a conformational change of AMP pyrophosphorylase induced by the binding of metabolites is a slow process as compared to the overall catalytic reaction. This hysteretic characteristic of AMP pyrophosphorylase may be one of the regulatory mechanisms in purine intermediary metabolism.