Computer-aided design (CAD) of Mn(II) complexes: superoxide dismutase mimetics with catalytic activity exceeding the native enzyme.

New Mn(II) macrocyclic pentaamine complexes derived from the biscyclohexyl-pyridine complex, M40403 ([manganese(II)dichloro[(4R,9R,14R,19R)-3,10,13,20,26-pentaazatetracyclo[20.3.1.0.(4,9)0(14,19)]hexacosa-1(26),-22(23),24-triene]]), are described here. The complex M40403 was previously shown to be a superoxide dismutase (SOD) catalyst with rates for the catalytic dismutation of superoxide to oxygen and hydrogen peroxide at pH = 7.4 of 1.2 x 10(+7) M(-1) s(-1).(1) The use of the computer-aided design paradigm reported previously for this class of Mn(II) complexes(2,3) led to the prediction that the 2S,21S-dimethyl derivative of M40403 should possess superior catalytic SOD activity. The synthesis of this new macrocyclic Mn(II) complex, [manganese(II)dichloro[2S, 21S-dimethyl-(4R,9R,14R,19R)-3,10,13,20,26-pentaazatetracyclo[20.3.1.0.(4,9)0(14,19)]hexacosa-1(26),22(23),24-triene]], 5, was accomplished via a high yield template condensation utilizing the linear tetraamine, N,N'-Bis[(1R,2R)-[2-(amino)]cyclohexyl]-1,2-diaminoethane, 1, 2,6-diacetylpyridine, and MnCl(2) to form the macrocyclic diimine complex, 2, which then is reduced. The two other possible dimethyl diastereomers of 5 (2R,21R-dimethyl,3, and 2R,21S-dimethyl, 6) were also prepared via reduction of the diimine complex 2. Two of these complexes, 3 and 5, were characterized by X-ray structure determination confirming their absolute stereochemistry as 2R,21R-dimethyl and 2S,21S-dimethyl, respectively. The results of the MM calculations which predict that the 2S,21S-dimethyl complex, 5, should be a high activity catalyst and that the 2R,21R-dimethyl complex, 3, should have little or no catalytic activity are presented. The catalytic SOD rates for these complexes are reported for each of these complexes and a correlation with the modeling predictions is established showing that 2R,21R-complex, 3, has no measurable catalytic rate, while the 2R,21S complex, 6, is identical to M40403, and the 2S,21S- complex, 5, possesses a very fast rate at pH = 7.4 of 1.6 x 10(+9) M(-1) s(-1) exceeding that of the native mitochondrial MnSOD enzymes.

Titration calorimetric analysis of AcylCoA recognition by myristoylCoA:protein N-myristoyltransferase.

Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme that catalyzes the transfer of myristic acid (C14:0) from myristoylCoA to the N-terminus of cellular proteins with a variety of functions. Nmts from an assortment of species display remarkable in vivo specificity for this rare acyl chain. To better understand the mechanisms underlying this specificity, we have used isothermal titration calorimetry as well as kinetic measurements to study the interactions of Nmt1p with acylCoA analogs having variations in chain length and/or conformation, analogs with alterations in the thioester bond, and analogs with or without a 3'-phosphate in their CoA moiety. MyristoylCoA binds to Nmt1p with a Kd of 15 nM and a large exothermic deltaH (-25 kcal/mol). CoA derivatives of C12:0-C16:0 fatty acids bind to Nmt1p with similar affinity, but with much smaller deltaH and a correspondingly less negative TdeltaS than myristoylCoA. Replacing the thioester carbonyl group with a methylene or removing the 3'-phosphate of CoA is each sufficient to prevent the low enthalpy binding observed with myristoylCoA. The carbonyl and the 3'-phosphate have distinct and important roles in chain length recognition over the range C12-C16. Acyltransferase activity parallels binding enthalpy. The naturally occurring cis-5-tetradecenoylCoA and cis-5,8-tetradecadienoylCoA are used as alternative Nmt substrates in retinal photoreceptor cells, even though they do not exhibit in vitro kinetic or thermodynamic properties that are superior to those of myristoylCoA. The binding of an acylCoA is the first step in the enzyme's ordered reaction mechanism. Our findings suggest that within cells, limitation of Nmt substrate usage occurs through control of acylCoA availability. This indicates that full understanding of how protein acylation is controlled not only requires consideration of the acyltransferase and its peptide substrates but also consideration of the synthesis and/or presentation of its lipid substrates.

Comparison of the reactivity of tetradecenoic acids, a triacsin, and unsaturated oximes with four purified Saccharomyces cerevisiae fatty acid activation proteins.

Saccharomyces cerevisiae contains at least five acyl-CoA synthetases (fatty acid activation proteins, or Faaps). Four FAA genes have been recovered to date. Recent genetic studies indicate that Faa1p and Faa4p are involved in the activation of imported fatty acids, while Faa2p activates endogenous pools of fatty acids. We have now purified Faa4p from S. cerevisiae and compared its fatty acid substrate specificity in vitro with the specificities of purified Faa1p, Faa2p, and Faa3p. Among C8-C18 saturated fatty acids, Faa4p and Faa1p both prefer C14:0. Surveys of C14 fatty acids with single cis-double bonds at C2-C12 indicated that Faa4p and Faa1p prefer Z9-tetradecenoic acid, although Faa4p's preference is much greater and also evident in C16 and C18 fatty acids. Faa4p's selectivity for fatty acids with a C9-C10 cis-double bond is a feature it shares with Faa3p and is notable since in yeast Ole1p, a microsomal cis-delta 9 desaturase, accounts for de novo production of monoenoic acyl-CoAs from saturated acyl-CoA substrates. Faa4p has no detectable acyl-CoA synthetase activity when incubated with tetradecenoic acids having a trans-double bond at C2-3, C4-5, C5-6, C6-7, C7-8, or C9-10. Faa3p can only use E9-tetradecenoic acid as a substrate, while E4-, E6- and E9-tetradecenoic acids can be used by Faa1p and Faa2p. E2-tetradecenoic acid is an Faap inhibitor, with Faa2p exhibiting the greatest sensitivity (IC50 = 2.6 +/- 0.2 microM). Triacsin C (1-hydroxy-3-(E,E,E,2',4',7'- undecatrienylidine)-1,2,3-triazene) has trans-double bonds at positions that correspond to those in E2-, E5-, and E7-tetradecenoic acids. This compound is a potent inhibitor of Faa2p (Ki = 15 +/- 1 nM; competitive with fatty acid), less potent against Faa4p (Ki = 2 microM), and not active against Faa1p or Faa3p (IC50 > 500 microM). Analysis of an n-tetradecanal plus a series of oximes (tridecanal oxime, 1-azadeca-1,3,5-trienol, and 1-azaundeca-1,3,5-trienol) indicated that the combination of an azenol moiety (R-CH = N-OH) plus adjacent unsaturation are critical for triacsin C's selective inhibition of Faa2p. Triacsin C and oxime derivatives appear to be very useful for defining differences in molecular recognition among S. cerevisiae acyl-CoA synthetases. The > 25,000-fold range in the inhibitory effects of triacsin C on these four Faaps suggests that it may be possible to develop other selective inhibitors of eukaryotic acyl-CoA synthetases.