Molecular enzymology of carnitine transfer and transport.

Carnitine (L-3-hydroxy-4-N-trimethylaminobutyric acid) forms esters with a wide range of acyl groups and functions to transport and excrete these groups. It is found in most cells at millimolar levels after uptake via the sodium-dependent carrier, OCTN2. The acylation state of the mobile carnitine pool is linked to that of the limited and compartmentalised coenzyme A pools by the action of the family of carnitine acyltransferases and the mitochondrial membrane transporter, CACT. The genes and sequences of the carriers and the acyltransferases are reviewed along with mutations that affect activity. After summarising the accepted enzymatic background, recent molecular studies on the carnitine acyltransferases are described to provide a picture of the role and function of these freely reversible enzymes. The kinetic and chemical mechanisms are also discussed in relation to the different inhibitors under study for their potential to control diseases of lipid metabolism.

N-(cyclohexanecarboxyl)-O-phospho-l-serine, a minimal substrate for the dual-specificity protein phosphatase IphP.

Three dual-specific phosphatases [DSPs], IphP, VHR, and Cdc14, and three protein-tyrosine phosphatases [PTPs], PTP-1B, PTP-H1, and Tc-PTPa, were challenged with a set of low molecular weight phosphoesters to probe the factors underlying the distinct substrate specificities displayed by these two mechanistically homologous families of protein phosphatases. It was observed that beta-naphthyl phosphate represented an excellent general substrate for both PTPs and DSPs. While DSPs tended to hydrolyze alpha-naphthyl phosphate at rates comparable to that of the beta-isomer, the PTPs PTP-1B and Tc-PTPa did not. PTP-H1, however, displayed high alpha-naphthyl phosphatase activity. Intriguingly, PTP-H1 also displayed much higher protein-serine phosphatase activity in vitro, 0.2-0.3% that toward equivalent tyrosine phosphorylated proteins, than did PTP-1B or Tc-PTPa. The latter two PTPs discriminated between the serine- and tyrosine-phosphorylated forms of two test proteins by factors of >/=10(4)-10(6). While free phosphoserine represented an extremely poor substrate for all of the DSPs examined, the addition of a hydrophobic "handle" to form N-(cyclohexanecarboxyl)-O-phospho-l-serine produced a compound that was hydrolyzed by IphP with high efficiency, i.e., at a rate comparable to that of free phosphotyrosine or p-nitrophenyl phosphate. VHR also hydrolyzed N-(cyclohexanecarboxyl)-O-phospho-l-serine (1 mM) at a rate approximately one-tenth that of beta-naphthyl phosphate. None of the PTPs tested exhibited significant activity against this compound. However, N-(cyclohexanecarboxyl)-O-phospho-l-serine did not prove to be a universal substrate for DSPs as Cdc14 displayed little propensity to hydrolyze it.

Stereoisomeric acylamidomorpholinium carnitine analogues: selective inhibitors of carnitine palmitoyltransferase I and II.

Acylamidomorpholinium carnitine analogues, 6-(tetradecanamidomethyl- and -hexadecanamidomethyl)-4,4-dimethylmorpholin-4-ium-2-a cetate, 1, synthesized as complete sets of stereoisomers, were assayed as inhibitors for isozymes of carnitine palmitoyltransferase (CPT). Microsomal CPT isoymes showed modest discrimination among the stereoisomers; while rat-liver mitochondrial CPT-I and CPT-II showed distinct differences. The tetradecanamidomethyl analogue of (2R,6S)-1 activated CPT-I but inhibited CPT-II.

Acylcarnitine analogues as topical, microbicidal spermicides.

Acylcarnitine analogues, (+)-6-Carboxylatomethyl-2-alkyl-4,4-dimethylmorpholinium (Z-n, where n = the number of carbons in the alkyl chain), synthesized in multi-gram quantities show in vitro activities as spermicides, anti-HIV agents, and inhibitors of the growth of Candida albicans. Activity improves with increasing chain length. Compound Z-15 is a candidate for further study as a topical, microbicidal spermicide.

Selective modulation of carnitine long-chain acyltransferase activities. Kinetics, inhibitors, and active sites of COT and CPT-II.

Carnitine acyltransferases in mitochondria, peroxisomes and the endoplasmic reticulum are different gene products and serve different metabolic functions in the cell. Here we summarize briefly evidence that carnitine octanoyltransferase (COT) from the peroxisomes and carnitine palmitoyltransferase II (CPT-II) from the mitochondria (both matrix facing enzymes) differ kinetically and demonstrate that they differ in their sensitivity to conformationally constrained inhibitors that mimic the reaction intermediate. Medium chain inhibitors are 15 times more effective on COT than on CPT-II and long chain inhibitors, such as hemipalmitoylcarnitinium, 80 times more effective on the mitochondrial enzyme. Thus, it may be possible to develop inhibitors to inhibit mitochondrial beta-oxidation with minimal effects on peroxisomal beta-oxidation and other acyl-CoA dependent reactions.

Change in the mode of inhibition of acetylcholinesterase by (4-nitrophenyl)sulfonoxyl derivatives of conformationally constrained choline analogues.

A chiral, five-step synthesis of 2-(hydroxymethyl)-2,4-dimethylmorpholine (12) from (R)- and (S)-2-methylglycidols gives an overall yield of 63%. Morpholines (R)- and (S)-12 are converted into 2-(azidomethyl)-2,4-dimethylmorpholine (15) via 2,4-dimethyl-2-[[(4-nitrophenyl)sulfonoxy]methyl]morpholine (14). The tertiary morpholines 12, 14, and 15 are quaternarized to afford 2-(hydroxymethyl)-2,4,4-trimethylmorpholinum iodide (2), 2,4,4-trimethyl-2-[[(4-nitrophenyl)sulfonoxy]methyl]morpholinium iodide (3), and 2-(azidomethyl)-2,4,4-trimethylmorpholinium iodide (4), respectively, which all inhibit acetylcholinesterase (AChE). These morpholinium inhibitors are compared with conformationally constrained aryl hemicholinium AChE inhibitors. Enantiomers of 2 and 4 are reversible competitive inhibitors of AChE, with values of Ki = 360 +/- 30 microM for (S)-2, 650 +/- 90 microM for (R)-2, 450 +/- 70 microM for (S)-4, and 560 +/- 30 microM for (R)-4, respectively. Enantiomers of 3 are noncompetitive inhibitors of AChE with values of Ki = 19.0 +/- 0.9 microM for (S)-3 and 50 +/- 2 microM for (R)-3, respectively. AChE shows a 2-fold chiral discrimination in the case of inhibition by 2 and 3. Inhibition also changes from competitive to noncompetitive when (3-hydroxyphenyl)-N,N,N-trimethylammonium iodide (18) [Ki = 0.21 +/- 0.06 microM; Lee, B. H., Stelly, T. C., Colucci, W. J., Garcia, J. G., Gandour, R. D., and Quinn, D. M. (1992) Chem. Res. Toxicol. 5, 411-418] is converted into [3-[(4-nitrophenyl)sulfonoxy]phenyl]-N,N,N-trimethylammonium iodide (5), Ki = 6.0 +/- 0.5 microM. These results indicate that the 4-nitrobenzenesulfonyl group controls the mode of inhibition.

2,6,6-Trimethyl-2-oxo-1,3-dioxa-6-azonia-2-phosphocyclooctane iodide.

The eight-membered ring in the title compound, C7H17NO3P+.I-, has a boat-chair conformation, with the local mirror plane passing through the cyclic O atom and methylene C atom adjacent to the N atom. The P = O bond is pseudo-axial and the P-CH3 bond is pseudo-equatorial. The P-N distance is 3.821 (2) A.

Methyl (2S,6S:2R,6R)-6-(2-cyanoethyl)-4,6-dimethyl-2-morpholineacetate.

The morpholine ring of the title compound, C12H20N2O3, adopts a chair conformation with an equatorial (methoxycarbonyl)methyl group. The cyanoethyl and (methoxycarbonyl)methyl groups are trans with respect to each other. The global minimum conformation, as computed by PCMODEL [Gajewski & Gilbert (1992). Molecular Modeling Package. Version 4.0], of the title compound agrees with that observed in the crystal. In the crystal, the torsion angles (N identical to)C-CH2-CH2-C(O), (N identical to C)CH2-CH2-C-O, O-CH-CH2-C(OOCH3) and (O)CH-CH2-C(O)-O(CH3) have values -170.0 (1), -45.9 (2), -71.6 (2) and 142.8 (1) degrees, respectively.

Comparison of the active sites of the purified carnitine acyltransferases from peroxisomes and mitochondria by using a reaction-intermediate analogue.

The carnitine acyltransferases contribute to the modulation of the acyl-CoA/CoA ratio in various cell compartments with consequent effects on many aspects of fatty acid metabolism. The properties of the enzymes are different in each location. The kinetic mechanisms and kinetic parameters for the carnitine acyltransferases purified from peroxisomes (COT) and from the mitochondrial inner membrane (CPT-II) were determined. Product-inhibition studies established that COT follows a rapid-equilibrium random-order mechanism, but CPT-II follows a strictly ordered mechanism in which acyl-CoA or CoA must bind before the carnitine substrate. Hemipalmitoylcarnitinium [(+)-HPC], a prototype tetrahedral intermediate analogue of the acyltransferase reaction, inhibits CPT-II 100-fold better than COT. (+)-HPC behaves as an analogue of palmitoyl-L-carnitine with COT. In contrast, with CPT-II(+)-HPC binds more tightly to the enzyme than do substrates or products, suggesting that it is a good model for the transition state and, unlike palmitoyl-L-carnitine, (+)-HPC can bind to the free enzyme. The data support the concept of three binding domains for the acyltransferases, a CoA site, an acyl site and a carnitine site. The CoA site is similar in COT and CPT-II, but there are distinct differences between the carnitine-binding site which may dictate the kinetic mechanism.

(R)-2-hydroxy-3-iodo-2-methylpropyl 4-nitrobenzenesulfonate.

The global minimum conformation, as computed by PCMODEL [Gajewski & Gilbert (1992). Serena Software, Bloomington, IN, USA], of the title compound agrees with that observed in the crystal. In the crystal, the torsion angles I--CH2--C--CH2O, ICH2--C--CH2--O, C--CH2--O--S and CH2--O--S--C are -57.7 (4), -61.7 (4), 171.2 (2) and -73.0 (3) degrees, respectively. Weak intermolecular hydrogen bonding connects OO--H and OS = O with an O ... O distance of 2.927 (4) A and an angle about the H atom of 165 (4) degrees.

(+)-Hemipalmitoylcarnitinium strongly inhibits carnitine palmitoyltransferase-I in intact mitochondria.

The reaction of the methyl ester of (R)-norcarnitine with 1-bromo-2-heptadecanone produces (+)-6-[(methoxycarbonyl)methyl]-2-pentadecyl-4,4-dimethylmorpholinium bromide, 3, which hydrolyzes to (+)-6-(carboxylatomethyl)-2-pentadecyl-4,4-dimethylmorpholinium (hemipalmitoylcarnitinium, HPC) upon treatment with aqueous sodium hydroxide. Single-crystal X-ray analyses have confirmed the structures of (+)-HPC and 3. (+)-HPC inhibits carnitine palmitoyltransferase (CPT-I) activity for the forward reaction (palmitoyl-CoA + carnitine-->) in intact mitochondria from rat heart and rat liver. (+)-HPC competitively (versus carnitine) inhibits CPT-I activity in both rat heart and liver mitochondria with Ki = 2.8 +/- 0.5 and 4.2 +/- 0.7 microM, respectively. As one of the strongest specific inhibitors of CPT-I, HPC is a potential therapeutic agent in myocardial ischemia and Type II diabetes.

2-hydroxy-4,4-dimethyl-2-(4-tolyl)-morpholinium bromide.

C13H20NO2+.Br-, M(r) = 302.2, monoclinic, P2(1)/c, a = 8.697 (2), b = 12.741 (3), c = 12.940 (2) A, beta = 103.39 (2) degrees, V = 1394.9 (8) A3, Z = 4, Dx = 1.439 g cm-3, lambda (Mo K alpha) = 0.71073 A, mu = 29.1 cm-1, F(000) = 624, T = 296 K, R = 0.038 for 1662 observations with I > 2 sigma (I) (of 2460 unique data). The morpholinium ring adopts the chair conformation with endocyclic torsion angle magnitudes 49.1 (4)-61.9 (4) degrees. The hydroxyl group is in the axial position of the morpholinium ring, with C--OH bond distance 1.401 (4) A. The hydroxy H atom points towards a Br ion; the interaction has O...Br distance 3.292 (2) A, H...Br distance 2.61 (3) A, and angle at H 160 (4) degrees.

Inhibition of acetylcholinesterase by hemicholiniums, conformationally constrained choline analogues. Evaluation of aryl and alkyl substituents. Comparisons with choline and (3-hydroxyphenyl)trimethylammonium.

2-Substituted-2-hydroxy-4,4-dimethylmorpholiniums (hemicholiniums) inhibit acetylcholinesterase (EC 3.1.1.7)-catalyzed hydrolysis of acetylthiocholine (ATCh). The 4-substituted arenes [NH2, NHC(O)CH3, Cl, CN, and NO2] have values of inhibition constants (Ki) that range from 220 to 3690 microM, which correlate with Hammett sigma, rho approximately 0.8. The alkyl compounds, hydrogen, methyl, tert-butyl, and trifluoromethyl, have values of Ki of 550, 560, 1200, and 1200 microM, respectively. These values compare favorably with Ki = 960 microM for choline. The conformation of AChE-bound choline must be gauche to support our suggestion that hemicholiniums are conformationally constrained analogues of choline. (3-Hydroxyphenyl)trimethylammonium (5) inhibits most strongly, Ki = 0.21 microM, of the compounds examined in this study. The solvent isotope effect (H2OKi/D2OKi = 0.83 +/- 0.04) suggests that inhibition by 5 involves hydrogen bonding. The binding by AChE of the hemicholiniums of various sizes and the strong binding of 5 support an earlier proposal [Schowen, K. B., Smissman, E. E., and Stephen, W. F., Jr. (1975) J. Med. Chem. 18, 292-300] that the active site of AChE has ample space for rotation about the C-C bond in choline. Compound 5, which has one more carbon between the hydroxy and trimethylammonium than does choline, inhibits much more potently than either choline or the hemicholiniums. Compound 5 provides a correct spacer to span the trimethylammonium recognition site and the esteratic site of AChE. This aromatic spacer interacts favorably with the hydrophobic active site, and the phenolic hydroxyl probably hydrogen bonds to the histidine in the esteratic site. Choline in any conformation and the hemicholiniums are too short to make a strong hydrogen bond.

Effect of carnitine acetyltransferase inhibition on rat hepatocyte metabolism.

Carnitine acetyltransferase (CAT) catalyzes the reversible transfer of short chain (less than six carbons in length) acyl groups from acyl-CoA thioesters to form the corresponding acylcarnitines. This reaction has been suggested to be of importance in decreasing cellular content of acyl-CoA under conditions characterized by accumulation of poorly metabolized, potentially toxic acyl-CoAs. To study the importance of the CAT reaction, the effect of CAT inhibitors on rat hepatocyte metabolism in the presence of propionate was examined. Acetyl-DL-aminocarnitine inhibited [14C]propionylcarnitine accumulation by isolated hepatocytes incubated with [14C]propionate (1.0-10.0 mM). Inhibition of propionylcarnitine formation by acetyl-DL-aminocarnitine was concentration dependent and was not due to non-specific cellular toxicity as [14C]glucose formation from [14C]propionate, and [1-14C]pyruvate oxidation were unaffected by the CAT inhibitor. Inhibition of propionylcarnitine formation was increased by preincubating hepatocytes with acetyl-DL-aminocarnitine, suggesting competition for cellular uptake between carnitine and the inhibitor. Hemiacetylcartinium (HAC) and meso-2,6-bis(carboxymethyl)4,4-dimethylmorpholinium bromide (CMDM), potent inhibitors of CAT in broken cell systems, did not inhibit hepatocyte propionylcarnitine formation under the conditions evaluated. Propionate (5 mM) inhibited hepatocyte pyruvate (10 mM) oxidation, and this inhibition was partially reversed by 5 mM carnitine. Addition of 5.0 mM acetyl-DL-aminocarnitine abolished the stimulatory effect of carnitine on pyruvate oxidation in the presence of propionate. These studies establish that acetyl-DL-aminocarnitine inhibits intact hepatocyte CAT activity, and thus provide a useful probe of the role of CAT in cellular metabolism. CAT activity appears to be critical for carnitine-mediated reversal of propionate-induced inhibition of pyruvate oxidation.

Intramolecular hemiacetals. The acid-base-catalyzed ring-chain interconversion of 2-substituted 2-hydroxy-4,4-dimethylmorpholinium cations in aqueous solution.

The ring-chain tautomerism in aqueous solution of some aryl-substituted morpholinium salts (bromides), has been studied and equilibrium constants are reported. In the crystals the substrates exist entirely in their cyclic forms as hemiacetals, but in aqueous solution NMR measurements reveal that an equilibrium is established between the cyclic (hemiacetal) and the noncyclic (ketone) form, the degree of ring-opening being more pronounced with electron-donating aryl substituents at the carbonyl carbon. The kinetics of the ring-chain interconversion in water has been investigated spectrophotometrically by a 'pH jump' stopped-flow technique. General base catalysis is observed with a Brønsted beta value apparently independent of substituent and equal to 0.60. The Hammett rho values for various base catalysts are close to those for very similar intermolecular reactions involving hemiacetal breakdown, leading to the suggestion of a 'normal' class n mechanism for base catalysis. For acid catalysis, however, a quite different situation is encountered, since no general acid but only (weak) catalysis by the hydronium ion can be detected. We believe this deviation from 'normal' general acid catalysis is caused by an electrostatic interaction, and we suggest that it might result from a change in the usual class e mechanism for general acid catalysis by a situation in which rate-limiting concerted proton transfer is replaced by rate-limiting preprotonation. This is supported by the observed drastic change in Hammett rho value for catalysis by the hydronium ion, compared with the 'normal' case. An interesting case is encountered for the 4-aminophenyl-substituted substrate, in which the amino group becomes protonated in acid solution, thus representing a new substituent. Despite this complication, the various equilibrium and rate constants may also be evaluated experimentally for this substrate.

Hydrogen bonding and conformational analysis of (R)-norcarnitine monohydrate.

(R)-4-(N,N-Dimethylammonio)-3-hydroxybutanoate [(R)-norcarnitine] monohydrate, C6H13NO3.H2O, Mr = 165.2, triclinic, P1, a = 5.9081 (5), b = 6.0438 (4), c = 6.9084 (7) A, alpha = 65.584 (6), beta = 81.957 (8), gamma = 77.771 (6) degrees, V = 219.1 (1) A3, Z = 1, Dx = 1.252 g cm-3, lambda(Cu K alpha) = 1.54184 A, mu = 8.46 cm-1, F(000) = 90, T = 296 K, R = 0.035 for 1763 observations (of 1772 unique data). Intermolecular hydrogen bonding dominates the structure. The molecule of water contacts three different zwitterions, with O...O distances 2.7767 (13), 2.720 (2) and 2.722 (2) A. A hydrogen bond between carboxylate and dimethylammonio links the zwitterions in head-to-tail motif, N...O 2.672 (2) A. Comparison with two other 4-ammonio-3-hydroxybutanoates reveals that the title compound adopts a different conformation along the backbone, N(+)--C--C(OH)--C--CO2-, with N(+)--C--C(OH)--C anti and C--C(OH)--C--CO2- gauche-.

1-Ethynyl-2,7-dimethoxynaphthalene: an example of hydrogen bonding between an ethynylic hydrogen and a methoxyl oxygen.

C14H12O2, Mr = 212.3, triclinic, P1, a = 6.6160 (12), b = 11.359 (2), c = 16.217 (2) A, alpha = 80.640 (11), beta = 86.305 (13), gamma = 78.858 (12) degrees, V = 1179.2 (4) A3, Z = 4, D chi = 1.196 g cm-3, lambda(Cu K alpha) = 1.54184 A, mu = 6.01 cm-1, F(000) = 448, T = 295 K, R = 0.039 for 3488 observations (of 4844 unique data). The crystal consists of two independent molecules related by a C--H...O contact. The donor is the ethynylic H on one molecule, and the acceptor is the methoxyl O ortho to the ethynylic group on the other molecule. The = C--H...O bond length (C to O distance) is 3.260 (2) A and the angle at H is 164 (1) degrees. The naphthalene ring system of the two independent molecules shows an average deviation from planarity of 0.007 (2) and 0.008 (2) A with respective maximum deviations of 0.015 (1) and 0.014 (2) A.

1-(1-chlorovinyl)-2,7-dimethoxynaphthalene.

C14H13ClO2, Mr = 248.71, monoclinic, P21/n, a = 11.291 (1), b = 7.343 (1), c = 15.223 (2) A, beta = 90.899 (8) degrees, V = 1262.0 (5) A3, Z = 4, Dx = 1.309 g cm-3, lambda(CU K alpha) = 1.54184 A, mu = 26.0 cm-1, F(000) = 520, T = 299 K, R = 0.041 for 2405 observations (of 2516 unique data). The average deviation from planarity is 0.019 (2) A with a maximum of 0.035 (1) A for the fused rings. The dihedral angle between the naphthalene system and the chlorovinyl group is 101.93 (4) degrees. The methoxy group ortho to the chlorovinyl adopts a conformation with the methyl group anti to the neighboring alpha carbon of the ring, with a C-C-O-C torsion angle of -175.6 (2) degrees. The other methoxy group has the methyl syn to the neighboring alpha carbon, with a C-C-O-C torsion angle of 1.9 (3) degrees.

Diisopropylammonium chloride.

C6H16N+.Cl-,Mr = 137.65, orthorhombic, P2(1)2(1)2(1), a = 7.825 (4), b = 8.257 (1), c = 13.268 (2) A, V = 857.3 (5) A3, Z = 4, Dx = 1.066 g cm-3, lambda(Mo K alpha) = 0.71073 A, mu = 3.6 cm-1, F(000) = 304, T = 295 K, R = 0.030 for 556 observations (of 901 unique data). Each H on the N atom is hydrogen bonded to a Cl atom. N--H...Cl bond lengths (N--Cl distance) are 3.180 (3) and 3.163 (3) A with N--H...Cl angles 176 (2) and 175 (2) degrees respectively. Each Cl atom is involved in two hydrogen bonds. The cation has an approximate twofold axis of symmetry.