Adenosyl Moiety Research Articles

The structural basis for substrate specificity and inhibition of human S-adenosylmethionine decarboxylase.

S-Adenosylmethionine decarboxylase belongs to a small class of amino acid decarboxylases that use a covalently bound pyruvate as a prosthetic group. It is an essential enzyme for polyamine biosynthesis and provides an important target for the design of anti-parasitic and cancer chemotherapeutic agents. We have determined the structures of S-adenosylmethionine decarboxylase complexed with the competitive inhibitors methylglyoxal bis(guanylhydrazone) and 4-amidinoindan-1-one-2'-amidinohydrazone as well as the irreversible inhibitors 5'-deoxy-5'-[N-methyl-N-[(2-aminooxy)ethyl]amino]adenosine, 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)amino]adenosine, and the methyl ester analogue of S-adenosylmethionine. These structures elucidate residues important for substrate binding and show how those residues interact with both covalently and noncovalently bound inhibitors. S-Adenosylmethionine decarboxylase has a four-layer alphabeta betaalpha sandwich fold with residues from both beta-sheets contributing to substrate and inhibitor binding. The side chains of conserved residues Phe7, Phe223, and Glu247 and the backbone carbonyl of Leu65 play important roles in binding and positioning the ligands. The catalytically important residues Cys82, Ser229, and His243 are positioned near the methionyl group of the substrate. One molecule of putrescine per monomer is observed between the two beta-sheets but far away from the active site. The activating effects of putrescine may be due to conformational changes in the enzyme, to electrostatic effects, or both. The adenosyl moiety of the bound ligand is observed in the unusual syn conformation. The five structures reported here provide a framework for interpretation of S-adenosylmethionine decarboxylase inhibition data and suggest strategies for the development of more potent and more specific inhibitors of S-adenosylmethionine decarboxylase.

Adenosine analogues as selective inhibitors of glyceraldehyde-3-phosphate dehydrogenase of Trypanosomatidae via structure-based drug design.

In our continuation of the structure-based design of anti-trypanosomatid drugs, parasite-selective adenosine analogues were identified as low micromolar inhibitors of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Crystal structures of Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, and human GAPDH's provided details of how the adenosyl moiety of NAD(+) interacts with the proteins, and this facilitated the understanding of the relative affinities of a series of adenosine analogues for the various GAPDH's. From exploration of modifications of the naphthalenemethyl and benzamide substituents of a lead compound, N(6)-(1-naphthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)adenosine (6e), N(6)-(substituted-naphthalenemethyl)-2'-deoxy-2'-(substituted-benzamido)adenosine analogues were investigated. N(6)-(1-Naphthalenemethyl)-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (6m), N(6)-[1-(3-hydroxynaphthalene)methyl]-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (7m), N(6)-[1-(3-methoxynaphthalene)methyl]-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (9m), N(6)-(2-naphthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)adenosine (11e), and N(6)-(2-naphthalenemethyl)-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (11m) demonstrated a 2- to 3-fold improvement over 6e and a 7100- to 25000-fold improvement over the adenosine template. IC(50)'s of these compounds were in the range 2-12 microM for T. brucei, T. cruzi, and L. mexicana GAPDH's, and these compounds did not inhibit mammalian GAPDH when tested at their solubility limit. To explore more thoroughly the structure-activity relationships of this class of compounds, a library of 240 N(6)-(substituted)-2'-deoxy-2'-(amido)adenosine analogues was generated using parallel solution-phase synthesis with N(6) and C2' substituents chosen on the basis of computational docking scores. This resulted in the identification of 40 additional compounds that inhibit parasite GAPDH's in the low micromolar range. We also explored adenosine analogues containing 5'-amido substituents and found that 2',5'-dideoxy-2'-(3,5-dimethoxybenzamido)-5'-(diphenylacetamido)adenosine (49) displays an IC(50) of 60-100 microM against the three parasite GAPDH's.

Role of reducing co-factors in catalytic reactions of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells

6-Hydroxymellein (6HM) synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, is capable of catalyzing the acyl-CoA condensation and the ketoreduction in the presence of the nucleotide reducing co-factors. Although free CoA at high concentrations functioned as the activator of the NADPH-dependent 6HM formation, the compound exhibited an appreciable inhibitory activity toward the reaction mediated by NADH. CoA showed a potent inhibitory activity against substrate entry into the reaction center of the NADH-associated enzyme while, in the presence of NADPH, the compound slightly inhibited the formation of the acylated enzyme. The catalytic rate of the synthase was appreciably decreased when NADPH was replaced by the deuterium-labeled compound, however, the kH/ kD value was markedly reduced if NADH and [D]NADH were employed as the reducing co-factors. These results suggest that the phosphate group attached to 2'-position of the adenosyl moiety of NADPH associated with the ketoreducing domain of 6HM synthase plays an important role in the regulation of the enzyme activity.

Structure-based design of submicromolar, biologically active inhibitors of trypanosomatid glyceraldehyde-3-phosphate dehydrogenase.

The bloodstream stage of Trypanosoma brucei and probably the intracellular (amastigote) stage of Trypanosoma cruzi derive all of their energy from glycolysis. Inhibiting glycolytic enzymes may be a novel approach for the development of antitrypanosomatid drugs provided that sufficient parasite versus host selectivity can be obtained. Guided by the crystal structures of human, T. brucei, and Leishmania mexicana glyceraldehyde-3-phosphate dehydrogenase, we designed adenosine analogs as tight binding inhibitors that occupy the pocket on the enzyme that accommodates the adenosyl moiety of the NAD+ cosubstrate. Although adenosine is a very poor inhibitor, IC50 approximately 50 mM, addition of substituents to the 2' position of ribose and the N6-position of adenosine led to disubstituted nucleosides with micromolar to submicromolar potency in glyceraldehyde-3-phosphate dehydrogenase assays, an improvement of 5 orders of magnitude over the lead. The designed compounds do not inhibit the human glycolytic enzyme when tested up to their solubility limit (approximately 40 microM). When tested against cultured bloodstream T. brucei and intracellular T. cruzi, N6-(1-naphthalenemethyl)-2'-(3-chlorobenzamido)adenosine inhibited growth in the low micromolar range. Within minutes after adding this compound to bloodstream T. brucei, production of glucose-derived pyruvate ceased, parasite motility was lost, and a mixture of grossly deformed and lysed parasites was observed. These studies underscore the feasibility of using structure-based drug design to transform a mediocre lead compound into a potent enzyme inhibitor. They also suggest that energy production can be blocked in trypanosomatids with a tight binding competitive inhibitor of an enzyme in the glycolytic pathway.

Role of reducing co-factor in cerulenin-insensitivity of 6-hydroxymellein synthase in carrot cell extract.

The activity of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme of carrot, was not inhibited by cerulenin in the presence of NADPH. However, cerulenin showed a marked inhibitory activity to the synthase if the reducing co-factor was omitted from the assay mixture. The synthase was also sensitive to the antibiotic even in the presence of NADPH when the acyl condensation site and the reducing domain at the reaction center of the enzyme were dissociated under the high ionic strength condition. In addition, the synthase activity was appreciably inhibited when NADH was employed instead of NADPH. These observations strongly suggest that a phosphate group attached to 2'-position of adenosyl moiety of NADPH molecule plays an important role in the apparent insensitivity of 6-hydroxymellein synthase toward cerulenin.

Selective tight binding inhibitors of trypanosomal glyceraldehyde-3-phosphate dehydrogenase via structure-based drug design.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the sleeping sickness parasite Trypanosoma brucei is a rational target for anti-trypanosomatid drug design because glycolysis provides virtually all of the energy for the bloodstream form of this parasite. Glycolysis is also an important source of energy for other pathogenic parasites including Trypanosoma cruzi and Leishmania mexicana. The current study is a continuation of our efforts to use the X-ray structures of T. brucei and L. mexicana GAPDHs containing bound NAD+ to design adenosine analogues that bind tightly to the enzyme pocket that accommodates the adenosyl moiety of NAD+. The goal was to improve the affinity, selectivity, and solubility of previously reported 2'-deoxy-2'-(3-methoxybenzamido)adenosine (1). It was found that introduction of hydroxyl functions on the benzamido ring increases solubility without significantly affecting enzyme inhibition. Modifications at the previously unexploited N6-position of the purine not only lead to a substantial increase in inhibitor potency but are also compatible with the 2'-benzamido moiety of the sugar. For N6-substituted adenosines, two successive rounds of modeling and screening provided a 330-fold gain in affinity versus that of adenosine. The combination of N6- and 2'-substitutions produced significantly improved inhibitors. N6-Benzyl (9a) and N6-2-methylbenzyl (9b) derivatives of 1 display IC50 values against L. mexicana GAPDH of 16 and 4 microM, respectively (3100- and 12500-fold more potent than adenosine). The adenosine analogues did not inhibit human GAPDH. These studies underscore the usefulness of structure-based drug design for generating potent and species-selective enzyme inhibitors of medicinal importance starting from a weakly binding lead compound.

Conformational studies of 5′-deoxyadenosyl-13-epicobalamin, a coenzymatically active structural analog of coenzyme B 12

5′-Deoxyadenosyl-13-epicobalamin(Ado-13-epiCbl) is a coenzymatically active structural analog of coenzyme B 12 (5′-deoxyadenosylcobalamin, AdoCbl) in which epimerization at the corrin ring C13 places the e propionamide side chain in an “upwardly” axial position. This would seem to bring the e side chain into direct steric contact with the adenosyl (Ado) moiety in its normal “southern” conformation (with the purine above the C ring) seen in the solid state for AdoCbl. Molecular mechanics calculations show that this conformation is the global minimum for AdoCbl, but that three other conformations represent local minima, including a “western”, a “northern”,and an “eastern” conformation, the last of which is known to be populated at room temperature from NMR studies. A structure for Ado-13-epiCbl has been generated by molecular dynamics/simulated annealing calculations starting from the neutron diffraction coordinates for AdoCbl after epimerization at C13. Molecular mechanics calculations on the resulting structure show that a “western” conformation is the global minimum, but that three other conformations, a “northern”, an “eastern”, and a “northeastern”, are of relatively low energy and separated by relatively low energy barriers. The traditional “southern” conformation does not appear as a local energy minimum and is some 6 kcal mol −1 destabilized relative to the global minimum due to geometric distortions to relieve steric strain. Two-dimensional homonuclear NMR experiments show extraordinary conformational flexibility in Ado-13-epiCbl with 21 NOE crosspeaks between Ado protons and non-Ado protons, 50% more than in AdoCbl itself. Together with the calculated geometries for the four Ado-13-epiCbl conformers, these data suggest that at least three, and possibly all four of the conformers are populated at room temperature, in contrast to AdoCbl, in which only the “southern” and “eastern” conformers are populated under these conditions. Considering the enzymology of Ado-13-epiCbl, the inaccessibility of its “southern” conformation, and the activity of other AdoCbl analogs structurally modified in the Ado moiety, it is suggested that it may well be the “eastern” conformation, accessible to both AdoCbl and Ado-13-epiCbl, which is enzymatically active.

Effect of phosphate residue of NADPH on the interaction between catalytic domains of a multifunctional polyketide synthetic enzyme 6-hydroxymellein synthase

Association of NADPH with the ketoreducing domain of 6-hydroxymellein synthase, a multifunctional polyketide synthetic enzyme of carrot, evoked the alternation of microstructure around the primary binding site of the co-substrates acetyl- and malonyl-CoAs, and this resulted in the marked decrease in K m value of the enzyme protein for acetyl-CoA. In contrast, the enzyme did not show the increase in the affinity to the substrate when NADPH was replaced by NADH. These results suggest that the phosphate residue attached to 2′-position of adenosyl moiety of NADPH molecule plays an important role in the co-operative interaction between these functional domains of the synthase.

Adenosylcobalamin-dependent glutamate mutase: examination of substrate and coenzyme binding in an engineered fusion protein possessing simplified subunit structure and kinetic properties.

Glutamate mutase is comprised of two weakly associating subunits; E and S, that combine to form the coenzyme binding site. The active holoenzyme assembles in a kinetically complex process in which both the stoichiometry and apparent Kd for adenosylcobalamin (AdoCbl) are dependent upon the relative concentrations of the two subunits, as is the enzyme's specific activity. To facilitate mechanistic and structural studies on this enzyme we have genetically fused the S subunit to the C-terminus of the E subunit through an 11 amino acid (Gly-Gln)5-Gly linker segment. This protein, GlmES, binds AdoCbl stoichiometrically and neither the affinity for AdoCbl nor the turnover number depends upon protein concentration. The kcat and Km for both substrate and coenzyme, together with the deuterium isotope effects on Vmax and Vmax/Km, have been determined for the GlmES-catalyzed reaction proceeding in both directions. Compared with wild type, the affinity for AdoCbl is unchanged, but for the conversion of L-glutamate to (2S,3S)-3-methylaspartate both kcat and Km for L-glutamate are decreased by about a third and the isotope effects are reduced, suggesting product release to be more rate-limiting. To test hypotheses concerning the activation of the coenzyme, we examined the binding of adenosylcobalamin, methylcobalamin, and cob(II)alamin to the enzyme. Each of these is bound with essentially the same affinity (2 microM), suggesting that, contrary to expectations, interactions between the protein and the adenosyl moiety do not serve to weaken the cobalt-carbon bond in the ground state.

The synthesis of the two S-adenosyl-methionine synthetases is differently regulated in Saccharomyces cerevisiae

S-adenosyl-L-methionine (AdoMet) is synthesized by transfer of the adenosyl moiety of ATP to the sulfur atom of methionine. This reaction is catalysed by AdoMet synthetase. In all eukaryotic organisms studied so far, multiple forms of AdoMet synthetases have been reported and from their recent study, it appears that AdoMet synthetase is an exceptionally well conserved enzyme through evolution. In Saccharomyces cerevisiae, we have demonstrated the existence of two AdoMet synthetases encoded by genes SAM1 and SAM2. Yeast, which is able to concentrate exogenously added AdoMet, is thus a particularly useful biological system to understand the role and the physiological significance of the preservation of two almost identical AdoMet synthetases. The analysis of the expression of the two SAM genes in different genetic backgrounds during growth under different conditions shows that the expression of SAM1 and SAM2 is regulated differently. The regulation of SAM1 expression is identical to that of other genes implicated in AdoMet metabolism, whereas SAM2 shows a specific pattern of regulation. A careful analysis of the expression of the two genes and of the variations in the methionine and AdoMet intracellular pools during the growth of different strains lead us to postulate the existence of two different AdoMet pools, each one supplied by a different AdoMet synthetase but in equilibrium with each other. This could be a means of storing AdoMet whenever this metabolite is overproduced, thus avoiding the degradation of a metabolite the synthesis of which is energetically expensive.

Lysine 2,3-Aminomutase: Support for a mechanism of hydrogen transfer involving S-adenosylmethionine

The conversion of L-lysine to L-beta-lysine is catalyzed by lysine 2,3-aminomutase. The reaction involves the interchange of the 2-amino group of lysine with a hydrogen at carbon 3. As such the reaction is formally analogous to adenosylcobalamin-dependent rearrangements. However, the enzyme does not contain and is not activated by this coenzyme. Instead it contains iron and pyridoxal phosphate and is activated by S-adenosylmethionine. Earlier experiments implicated adenosyl-C-5' of S-adenosylmethionine in the hydrogen transfer mechanism, apparently in a role similar or analogous to that of adenosyl moiety of adenosylcobalamin in the B12-dependent rearrangements. The question of whether both hydrogens or only one hydrogen at adenosyl-C-5' participate in the hydrogen-transfer process has been addressed by carrying out the lysine 2,3-aminomutase reaction with S-[5'-3H] adenosylmethionine in the presence of 10 times its molar concentration of enzyme. Under these conditions all of the tritium appeared in lysine and beta-lysine, showing that C-5'-hydrogens participate. To determine whether hydrogen transfer is compulsorily intermolecular and intramolecular, various molar ratios of [3,3-2H2]lysine and unlabeled lysine were submitted to the action of lysine 2,3-aminomutase under conditions in which 10-15% conversion to beta-lysine occurred. Mass spectral analysis of the beta-lysine for monodeutero and dideutero species showed conclusively that hydrogen transfer is both intramolecular and intermolecular. The results quantitatively support our postulate that activation of the enzyme involves a transformation of S-adenosylmethionine into a form that promotes the generation of an adenosyl-5' free radical, which abstracts hydrogen from lysine to form 5'-deoxyadenosine as an intermediate.

Arsenate metabolism in aquatic plants

AbstractOf the several types of arsenic metabolic pathways in algae and aquatic higher plants, production of arsenoribosides is predominant and most interesting. Insertion of the ribosyl or adenosyl moiety by transfer from S‐adenosylmethioninine or possibly from adenosylcobalamin is the critical biochemical step which, as yet, has not been experimentally demonstrated. Oceanic or other environmental arsenate (AsO43−), absorbed in the plant's quest for phosphate, is fixed by reaction with ATP to yield the phosphoric arsenic anhydride APAs, a short‐lived but reducible intermediate which is converted to arsenic(III). The Hill Reaction, or thioredoxin reductase, reduces it to an arsine oxide (HAsO) or arsonous acid [HAs(OH)2] depending upon the water content of its environment. This readily diffusible reagent avidly attacks sulfhydryl groups of proteins to produce arsonous thioesters. The Sargassum group of algae appears to process arsenate no further than this. The reduced arsenic may be freed from its sulfur bondage by reaction with 2,3‐dimercaptopropan‐1‐ol (BAL) or dithiothreitol. In experiments with Sargassum fluitans and Sargassum natans no arsenoribosides were observed. Only protein‐bound arsenic was observed. It could be liberated by trituration with dithiothreitol to produce the cyclic arsonous dithioester.Most diatoms, dinoflagellates and macroalgae as well as freshwater higher plants release such protein‐bound arsenic as a result of sequential methylation and adenosylation. Ultimately the products are trialkylarsine oxides, innocuous substances which are slowly or not‐at‐all metabolized by herbivorous animals or bacteria. Fortunately mammals and most animals also excrete the arsenoribosides readily, unchanged. Arsenic metabolism by a cyanobacterium, Phormidium sp., was described by Matsuto et al. (Comp. Biochem. Physiol., 1984, 78c:377) as involving two modes of arsenate fixation, reduction, and excretion. We have extended those experiments with Phormidium persicinum. We have analyzed algae from 2‐ and 7‐day culture in radioarsenate media. The arsenic products included 80% of arsenolipid, similar if not identical to that formed in the brown algae. The water‐soluble products were in low concentration. Insoluble, protein‐bound arsonous thioesters accounted for 8% of the fixed arsenic. The mechanism of arsenic depuration in Phormidium appears to be primarily lipid‐mediated.

On the structural specificity of puromycin binding to Escherichia coli ribosomes.

We have examined the structural specificity of the puromycin binding sites on the Escherichia coli ribosome that we have previously identified [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry 19, 3809-3817, and references cited therein] by examining the interactions of a series of adenine-containing compounds with these sites. We have used as measures of such interactions the inhibition of [3H]puromycin photoincorporation into ribosomal proteins from these sites, the site-specific photoincorporation of the 3H-labeled compounds themselves, and the inhibition of peptidyl transferase activity. For the first two of these measures we have made extensive use of a recently developed high-performance liquid chromatography (HPLC) method for ribosomal protein separation [Kerlavage, A. R., Weitzmann, C., Hasan, T., & Cooperman, B.S. (1983) J. Chromatogr. 266, 225-237]. We find that puromycin aminonucleoside (PANS) contains all of the structural elements necessary for specific binding to the three major puromycin binding sites, those of higher affinity leading to photoincorporation into L23 and S14 and that of lower affinity leading to photoincorporation into S7. Although tight binding to the L23 and S7 sites requires both the N6,N6-dimethyl and 3'-amino groups within PANS, only the N6,N6-dimethyl group and not the 3'-amino group is required for binding to the S14 site. Our current results reinforce our previous conclusion that photoincorporation into L23 takes place from the A' site within the peptidyl transferase center and lead us to speculate that the S14 site might be specific for the binding of modified nucleosides. They also force the conclusion that puromycin photoincorporation proceeds through its adenosyl moiety.

Inactivation of dioldehydrase in the presence of a coenzyme-B 12 analog

We have previously shown that a coenzyme-B 12 analog, adenosylcobalamin (AdoCbl)-(e-OH), with the e-propionamide group converted to a carboxylic acid, serves as a poor coenzyme for dioldehydrase. During the course of the catalytic process, the enzyme AdoCbl-(e-OH) complex becomes catalytically inactive ( T. Toraya, E. Krodel, A. S. Mildvan, and R. H. Abeles, 1979, Biochemistry 18, 417–426 ). We have now examined the mechanism of this inactivation further. Inactivation only occurs in the presence of substrate. The dioldehydrase coenzyme analog complex is stable in the absence of substrate. In the inactivated complex, the coenzyme analog was stoichiometrically converted to a cob(II)alamin species. The cob-(II)alamin formed remained irreversibly bound at the active site of the enzyme and resisted oxidation by O 2 even in the presence of CN −. Stoichiometric formation of 5′-deoxyadenosine from the 5′-deoxy-5′-adenosyl moiety of the coenzyme analog was demonstrated with [8- 14C]-AdoCbl(e-OH). This nucleoside also remained tightly bound to the enzyme and was not exchangeable with free 5′-deoxyadenosine nor was it removed by Sephadex chromatography. The rate of inactivation showed no deuterium isotope effect when the inactivation occurred in the presence of l,2-propanediol-l- d 2. The inactivated complex was resolved by acid ammonium sulfate treatment into the intact apoenzyme and the hydroxocobalamin derivative. This indicates that the apoenzyme itself is not modified in the inactivation process. These results suggest that the inactivation reaction occurs from one of the intermediates in the normal catalysis. We propose that the inactivation is due to incorrect binding of the modified coenzyme in an intermediate of the catalytic process. This incorrect binding leads to the loss of the substrate radical, and consequently, to loss of catalytic activity.

Studies of the ribosome-associated vitamin B 12s adenosylating enzyme of Lactobacillus leichmannii

The ribosomes of Lactobacillus leichmannii (ATCC 7830) are the loci of an enzyme system that converts vitamin B 12 (cyanocobalamin, CN-Cbl) to adenosylcobalamin (AdoCbl) in two steps, reduction of B 12a (Co III) to B 12s (Co I) by B 12a reductase and the adenosylation of B 12s to AdoCbl by an adenosylating enzyme. The vitamin B 12 reductase, as in other organisms, is unstable. Adenosylating enzyme, however, is readily demonstrable. Reported experiments deal primarily with that enzyme. Evidence of the association between ribosomes and adenosylating enzyme was found in sucrose density gradient analyses. Intact, washed ribosomes yielded an enzyme activity profile that coincided with the ultraviolet maximum of 70S reference ribosomes. When intact ribosomes were exposed to 2.5 m CsCl so that 70% of ribosomal protein was recoverable in the 144,000 g supernatant fraction and >90% of RNA was in the pellet, adenosylating enzyme was found in the supernatant fraction. “Stripped” ribosomes had low levels of enzyme activity and could reassociate with free enzyme protein. Stripped ribosomes remained competent in protein synthesis. Hence, adenosylating enzyme is not an integral ribosome component. Partially purified ribosome-associated B 12s adenosylating enzyme has requirements for vitamin B 12s, ATP, and Mn 2+, though Mn 2+ could be partially replaced by Mg 2+. Isotopic studies showed that ATP is the source of the adenosyl moiety of AdoCbl and that inorganic tripolyphosphate is a reaction product. Substantial adenosylating activity is associated with ribosomes only in the vitamin B 12-requiring lactobacilli, L. delbrueckii (ATCC 9649) and L. leichmannii. Surprisingly, L. casei (ATCC 9595) ribosomes displayed a measurable, if low, level of activity. L. acidophilus (ATCC 11506) ribosomes had no detectable activity. The bulk of the activity in Clostridium tetanomorphum (ATCC 3606) and Propionibacterium shermannii (ATCC 9614) is in the 144,000 g supernatant fraction. Ribosomes from animal cells (liver, reticulocytes, and Ehrlich ascites tumor) were without detectable activity.

Benzoyl and Hydroxybenzoyl Esters of Coenzyme A: PURIFICATION AND NUCLEAR MAGNETIC RESONANCE CHARACTERIZATION; CONFORMATION IN SOLUTION

Abstract Benzoyl-CoA and o-, m-, and p-hydroxybenzoyl-CoA were synthesized by reacting the benzoyl chloride (formed from the respective benzoic acid and oxalyl chloride) with reduced CoA. The products were purified by chromatography (Sephadex LH 20, ethanol-water, 40:60 (v/v) and treatment with Chelex. Purity was confirmed in several ways: (a) a single ultraviolet-absorbing spot with a unique Rf value was observed for each of the CoA esters in each of several thin layer chromatographic solvent systems; (b) elemental stoichiometry (C, N, P, S, Li, H) agreed with prediction; (c) the values of ultraviolet extinction coefficients (emm) obtained by dry weight, enzymic release of thiol, and comparison of additive spectra of CoA and the respective benzoate with esterolysis spectra of the benzoylCoA all agreed (see accompanying article (18)); (d) the proton magnetic resonance (PMR) spectra were consistent with the structures of the benzoyl-CoAs and contained no extraneous signals. All of the signals in the PMR spectra of CoA, of acetyl-CoA and of the benzoyl-CoAs were assigned to specific moieties of the respective molecules. A downfield displacement of the CH2-S signal relative to its position in the spectrum of reduced CoA is characteristic of substitution on the sulfur atom, e.g. oxidized CoA, acetyl-CoA, benzoyl-CoA. All other signals of the CoA moiety in spectra of reduced CoA and acetyl-CoA under various conditions have identical chemical shifts. In contrast, in spectra of each of the benzoyl-CoAs in D2O, the (C-1')-H, (C-2)-H and (C-8)-H signals of the adenosyl moiety and the signals of the respective benzoyl protons are displaced upfield with respect to their positions in spectra of equimolar admixtures of reduced CoA with the respective benzoate (all other signals (except CH2-S) are essentially unchanged). These differences in chemical shift (Δδ) are independent of benzoyl-CoA concentration. The Δδ values are decreased by increased temperature and there is no difference between the spectrum of benzoyl-CoA in 70 % methanol-d4 (30 % D2O) and that of the CoA-benzoate admixture in D2O. These PMR data, and concentration, temperature, and solvent data suggested a specific intramolecular complex involving a ring stacking interaction between the adenosyl and benzoyl moieties with the charged and polar groups of the phosphopantetheine moiety facing the exterior, and the methyl and methylene groups forming a hydrophobic interior of the folded molecule. A Corey-Pauling-Koltun molecular model of the folded benzoyl-CoA molecule was constructed which allowed direct measurement of the distances of the three adenosyl protons from the center of the benzene ring. The values of Δδ predicted from these distances (due to the benzene ring current effect) correlated well with those observed. The values of ΔG = -0.7 Cal per mole, ΔH = -8.9 Cal per mole, and ΔS = -27 e.u. calculated from the temperature-dependence data are consistent with the proposed model of the intramolecular complex, involving more than one type of weak interaction. It is noted that approximately 65 % of the molecules of benzoyl-CoA exist in the folded conformation near physiological temperature.

The Mechanism of Action of Ethanolamine Ammonia-Lyase, a B12-dependent Enzyme: THE REVERSIBLE FORMATION OF 5′-DEOXYADENOSINE FROM ADENOSYLCOBALAMIN DURING THE CATALYTIC PROCESS

Abstract When l-2-aminopropanol is added to a reaction mixture containing ethanolamine ammonia-lyase and adenosylcobalamin, the carbon-cobalt bond of adenosylcobalamin is rapidly cleaved. The adenosyl moiety of the cofactor accumulates as 5'-deoxyadenosine, or a compound which readily converts to 5'-deoxyadenosine, while the cobalamin moiety is converted to a species with a spectrum resembling that of cob(II)alamin. No loss in enzymatic activity occurs. The presence of 5'-deoxyadenosine, therefore, cannot be associated with inactivation of the enzyme. The formation of 5'-deoxyadenosine from adenosylcobalamin is reversible. This provides further evidence for intermediate involvement of the nucleoside in the catalytic process. Upon longer incubation with l-2-aminopropanol, the enzyme-adenosylcobalamin complex losses its activity, while its spectrum changes to that of hydroxocobalamin. Upon denaturation, 5'-deoxyadenosine is again isolated. We tentatively attribute this inactivation to the decomposition of a (hypothetical) intermediate substrate-cobalamin adduct.

The photolysis of 5′-deoxyadenosylcobalamin under anaerobic conditions

Nuclear magnetic resonance and mass spectrometry have been used to confirm that the nucleoside product formed when 5′-deoxyadenosylcobalamin is photolysed under anaerobic conditions is 8,5′-cyclic adenosine. The synthesis of 5′-deoxyadenosylcobalamin, which is tritiated at C 8 of the adenosyl moiety, provides the confirmation that the photolysis mechanism involves a reaction between the 5′-methylene carbon radical and C 8 of the adenine ring.

Inhibition of ATP: l-methionine S-adenosyltrasferase of bakers' yeast by structural analogues of ATP

Highly purified preparations of ATP: l-methionine S-adenosyltransferase (EC 2.5.1.6) of bakers' yeast display a virtually completely specific requirement for ATP. Kinetics analyses of the binding of a number of structural analogues and components of ATP have permitted assessment of the importance of various regions of the ATP molecular for “binding” and for “recognition” of the nucleotide. Adenosine or deoxyadenosine have almost no affinity for the enzyme, their mono- and dipphosphates are very weak inhibitors competitive with respect to ATP. Various nucleoside triphosphates are more powerful inhibitors competitive with respect to ATP and non-competitive with respect to l-methionine. Tripolyphosphate is an excellent inhibitor ( K i = 0.05 mM) and tetraphosphate also inhibits the enzyme ( K i = 0.18 mM). The following phosphonate analogues of ATP are powerful inhibitors: α,β-methylene-ATP ( K i = 0.55 mM); β,γ-methylene-ATP ( K i = 2.19 mM) and α,β-methylene-adenosine tetraphosphate ( K i = 0.17 mM). All of these phosphonates compete with ATP and the K i values refer to this competition when l-methionine approaches saturation. It is concluded from kinetic studies with adenosine and its analogues, that the triphosphate moiety of ATP is of primary importance for the binding of the substrate to the enzyme, while the adenosyl group of ATP is of primary importance for the substrate recognition, but with low affinity for the enzyme. Kinetic patterns obtained with analogues of ATP as inhibitors are consistent with the previous proposition that the ATP: l-methionine S-adenosyltransferase reaction involves a random-ordered bi-mechanism. The results of the present studies also suggest that the adenosyl group remains enzyme-bound prior to its binding to the sulfur atom of l-methionine, and that the triphosphate site-directed recognition of the adenosyl moiety enables ATP to gain virtually absolute substrate specificity.

Electron Spin Resonance Studies with Dioldehydrase: EVIDENCE FOR RADICAL INTERMEDIATES IN REACTIONS CATALYZED BY COENZYME B12

Abstract When 1,2-propanediol is added to dioldehydrase-coenzyme B12, electron spin resonance signals appear which indicate the presence of at least two paramagnetic species. One signal, at g = 2.2, is attributed to Co(II) of B12(r). Other signals, at g ≈ 2.0, which show different power saturation behavior, are tentatively assigned to organic radicals, possibly derived from the substrate and the adenosyl moiety of the coenzyme. The occurrence of such species is in accord with a previously proposed mechanism (Finlay, T. H., Valinsky, J., Sato, K., and Abeles, R. H. (1972) J. Biol. Chem. 247, 4197–4207). Integration of the signal indicates that approximately 0.75 unpaired electrons are present per molecule of enzyme-coenzyme complex. Signals with similar but not identical g values were obtained with the substrate analogues chloroacetaldehyde and glycolaldehyde. With chloroacetaldehyde the signal at g = 2.0 shows pronounced fine structure and non-uniform saturation behavior, confirming the multiplicity and substrate dependence of this signal. With both substrate analogues nearly two unpaired electrons are detected per molecule of enzyme-coenzyme complex. The paramagnetic species have only a small effect on the longitudinal relaxation rate of the water protons, suggesting that they are shielded from the solvent. The distance of closest approach of the water protons to the paramagnetic center was estimated to be 10.2 A. These experiments established that dioldehydrase can bring about the homolytic cleavage of the carbon-cobalt bond of coenzyme B12 and suggest the possibility that radical species may be intermediates in the catalytic reaction. A kinetic analysis will be required to establish conclusively that the radicals play a significant role in the catalytic process.