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(1) During peroxisomal beta-oxidation of [U-14C]hexadecanoate, at concentrations higher than 100 microM, long-chain 3-oxoacyl-CoA-esters and 3-oxobutyryl-CoA accumulate. Only 3-oxobutyryl-CoA accumulates at a low concentration of [U-14C]hexadecanoate. Accumulation of long chain 3-oxoacyl-CoA esters is most extensive when the supply of CoA can be considered limiting for beta-oxidation. (2) Added acetyl-CoA was found to inhibit peroxisomal beta-oxidation. This inhibition was not significantly relieved by added L-carnitine and carnitine acetyltransferase (EC 2.3.17). (3) Added L-carnitine, at concentrations below 0.2 mM, was found to stimulate peroxisomal beta-oxidation of [U-14C]hexadecanoate by up to 20%, causing the conversion of acetyl-CoA into acetylcarnitine. Higher concentrations of L-carnitine were progressively inhibitory to beta-oxidation. This effect was specific for L-carnitine as both D-carnitine and aminocarnitine neither caused stimulation at low concentrations, nor inhibition at higher concentrations. Added L-carnitine caused accumulation of acylcarnitines of chain-lengths ranging from 4 to 16 carbon-atoms. The inhibition observed with higher concentrations of added L-carnitine is likely due to conversion of [U-14C]hexadecanoate into [U-14C]hexadecanoylcarnitine. (4) Low concentrations of added hexadecanoylcarnitine was shown to inhibit peroxisomal beta-oxidation by about 15%, while added acetylcarnitine did not inhibit at concentrations up to 100 microM. (5) These data are interpreted to indicate significant control being exerted on flux at the stage of thiolysis either directly by means of CoA availability, or indirectly by means of the rate of acetyl-CoA generation.

A study of the biosynthesis of coenzyme A (CoA), a critical cofactor in the metabolism of lipids and other molecules in higher plants, was initiated. Pantothenate kinase was partially purified from spinach leaves. This enzyme was predominantly localized in the chloroplast with very little activity observed in the mitochondria or cytosol. DEAE-agarose chromatography resolved two pantothenate kinase activity peaks which differed in their requirement for reductant, stability upon boiling, and reactivity in the presence of spinach holo-acyl carrier protein (ACP) I. One active peak of this enzyme was further purified on Cibacron blue 3GA to yield a preparation containing pantothenate kinase enriched to 20% of the total protein within the fraction. Pantothenate kinase was inhibited by malonyl-CoA, but not by CoASH or acetyl-CoA, and the activity was stabilized by the phosphatase inhibitors sodium molybdate, sodium tungstate, and the phosphatase substrate glycerol 2-phosphate, but was inhibited by sodium fluoride. Further experiments demonstrated a linear increase in pantothenate kinase activity during spinach seed germination, consistent with a role for this enzyme in the developmental utilization of seed triacylglycerol.

The inhibition of carnitine palmitoyltransferase (CPT, EC 2.3.1.21) by malonyl-CoA, acetyl-CoA and free CoA was studied in sonicated skeletal-muscle homogenates from normal human subjects and from five patients with a mutant CPT [Zierz & Engel (1985) Eur. J. Biochem. 149, 207-214]. (1) Malonyl-CoA, acetyl-CoA and CoA were competitive inhibitors of CPT with palmitoyl-CoA. (2) Acetyl-CoA and CoA inhibited normal and mutant CPT to the same degree, whereas malonyl-CoA inhibited mutant CPT more than normal CPT. (3) Triton X-100 abolished the inhibition of normal CPT by malonyl-CoA, but not by acetyl-CoA or CoA. Triton X-100 by itself caused loss of activity of the mutant CPT. (4) In the concentration range 0.1-0.4 mM, the inhibitory effects of any two of the three inhibitors were synergistic. (5) The inhibitory constants (Ki) for acetyl-CoA and CoA were close to 45 microM. The Ki for malonyl-CoA was 200-fold lower, or 0.22 microM. Addition of 40 microM-acetyl-CoA or CoA resulted in a 3-fold increase in the Ki for acetyl-CoA. Addition of 20 microM-CoA resulted in a 3-fold increase in the Ki for acetyl-CoA. (6) The findings indicate that acetyl-CoA and CoA can inhibit CPT at the catalytic site or a nearby site which is different from that at which malonyl-CoA inhibits CPT. (7) The fact that small changes in the concentration of acetyl-CoA and CoA can antagonize the inhibitory effect of malonyl-CoA suggests that these compounds could modulate the inhibition of CPT by malonyl-CoA.

Rat mammary gland acetyl-CoA carboxylase (acetyl-CoA:carbon dioxide ligase (ADP forming), EC 6.4.1.2) is rapidly and irreversibly inactivated by micromolar concentrations of S-(4-bromo-2,3-dioxobutyl)-CoA (BDB-CoA) or p-hydroxymercuribenzoate (PHMB). Inhibition of both half reactions (i.e., the biotin carboxylation and the carboxyltransferase) catalyzed by acetyl-CoA carboxylase closely parallels loss in overall activity (malonyl-CoA synthesis). The presence of a substrate or product (acetyl-CoA, ATP, ADP, Pi) or inhibitor (palmitoyl-CoA) does not protect the enzyme from inhibition caused by BDB-CoA or PHMB. On the other hand, citrate, an activator of acetyl-CoA carboxylase, affords substantial protection against inhibition by BDB-CoA and PHMB. Covalent modification by BDB-CoA or PHMB appears to lock acetyl-CoA carboxylase in an inactive conformation (15-30 S) that is unable to undergo citrate-induced self-association into the catalytically competent polymeric form.

The translocation of adenine nucleotides across the inner mitochondrial membrane and the tissue concentration of long-chain acyl-CoA esters were studied in dog heart after experimental myocardial ischemia. Ligation of the anterior coronary artery initiated events leading to an early decrease in adenine nucleotide translocase activity. A reciprocal increase in the concentration of heart tissue long-chain acyl-CoA esters was also observed. Adjacent nonischemic tissue showed changes intermediate between that of ischemic and normal heart tissue. It is postulated that a decrease in fatty acid oxidation after myocardial ischemia would lead to an accumulation of long-chain acyl-CoA esters, which in turn would inhibit adenine nucleotide translocation. The net result would be a lowering of the energy charge of the cell, adversely affecting muscle contraction and electrical conduction.

Deviations from Michealis-Menten kinetics in the pig-heart citrate synthase (citrate-oxaloacetate-lyase(pro-3S-CH2-COO-leads to acetyl-CoA), EC 4.1.3.7) system have been characterized and analyzed in view of the kinetic theory described in the preceding paper. The enzymic condensation reaction between acetyl-CoA and oxaloacetate is subject to substrate-inhibition by acetyl-CoA. This can be attributed to the formation of a productive enzyme-acetyl-CoA complex with a dissociation constant of 110 uM. The binding of acetyl-CoA to the enzyme decreases the on-velocity constant for oxaloacetate-binding from 4000 min-1- micrometer-1 to 1700 min-1-micrometer-1. The affinity of citrate synthase for oxaloacetate increase at least 20-fold on the binding of acetyl-CoA. The latter cooperativity effect can be attributed to a more than 45-fold decrease of the off-velocity constant for oxaloacetate-binding.