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Evidence of a preferred kinetic pathway in the carnitine acetyltransferase reaction.

ABSTRACT: Mammalian carnitine acetyltransferase (CrAT) is a mitochondrial enzyme that catalyzes the reversible transfer of an acetyl group from acetyl-CoA to carnitine. CrAT knockout studies have shown that this enzyme is critical to sustain metabolic flexibility, or the ability to switch between different fuel types, an underlying theme of the metabolic syndrome. These recent physiological findings imply that CrAT dysfunction, or its catalytic impairment, may lead to disease. To gain insight into the CrAT kinetic mechanism, we conducted stopped-flow experiments in various enzyme substrate/product conditions and analyzed full progress curves by global fitting. Simultaneous mixing of both substrates with CrAT produced relatively fast kinetics that follows an ordered bi bi mechanism. A great preference for ordered binding is supported by stopped-flow double mixing experiments such that premixed CrAT with acetyl-CoA or CoA demonstrated a biphasic decrease in initial rate that produces about a 100-fold attenuation in catalysis. Double mixing experiments also revealed that the CrAT initial rate is inhibited by 50% in approximately 8 s by either acetyl-CoA or CoA premixing. Analysis of available CrAT structures support a substrate conformational change between acetyl-CoA/CoA binary versus ternary complexes. Additional viscosity-based kinetic experiments yielded strong evidence that product release is the rate limiting step in the CrAT-catalyzed reaction.

SUBMITTER: Kratochvil MJ

PROVIDER: S-EPMC7484016 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Some kinetic studies on the mechanism of action of carnitine acetyltransferase.

Project description:1. Michaelis constants for substrates of carnitine acetyltransferase have been shown to be independent of the concentration of second substrate present. This applies to the forward reaction between acetyl-l-carnitine and CoASH, and to the back reaction between l-carnitine and acetyl-CoA. 2. Product inhibition of both forward and back reactions has been studied. Evidence has been obtained for independent binding sites for l-carnitine and CoASH. Acetyl groups attached to either substrate occupy overlapping positions in space when the substrates are bound to the enzyme. 3. Possible reaction mechanisms involving the ordered addition of substrates have been excluded by determining kinetic constants in the presence and absence of added product. 4. d-Carnitine and acetyl-d-carnitine have been shown to inhibit competitively with respect to l-carnitine and acetyl-l-carnitine. 5. It is concluded that the mechanism of action of carnitine acetyltransferase involves four binary and two or more ternary enzyme complexes in rapid equilibrium with free substrates, the interconversion of the ternary complexes being the rate-limiting step. The possible intermediate formation of an acetyl-enzyme cannot be excluded, but this could only arise from a ternary complex.

| S-EPMC1264953 | biostudies-other

Obesity and lipid stress inhibit carnitine acetyltransferase activity.

Project description:Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes.

| S-EPMC3966698 | biostudies-literature

Carnitine Acetyltransferase Mitigates Metabolic Inertia and Muscle Fatigue during Exercise.

Project description:Acylcarnitine metabolites have gained attention as biomarkers of nutrient stress, but their physiological relevance and metabolic purpose remain poorly understood. Short-chain carnitine conjugates, including acetylcarnitine, derive from their corresponding acyl-CoA precursors via the action of carnitine acetyltransferase (CrAT), a bidirectional mitochondrial matrix enzyme. We show here that contractile activity reverses acetylcarnitine flux in muscle, from net production and efflux at rest to net uptake and consumption during exercise. Disruption of this switch in mice with muscle-specific CrAT deficiency resulted in acetyl-CoA deficit, perturbed energy charge, and diminished exercise tolerance, whereas acetylcarnitine supplementation produced opposite outcomes in a CrAT-dependent manner. Likewise, in exercise-trained compared to untrained humans, post-exercise phosphocreatine recovery rates were positively associated with CrAT activity and coincided with dramatic shifts in muscle acetylcarnitine dynamics. These findings show acetylcarnitine serves as a critical acetyl buffer for working muscles and provide insight into potential therapeutic strategies for combatting exercise intolerance.

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Carnitine acetyltransferase: A new player in skeletal muscle insulin resistance?

Project description:Carnitine acetyltransferase (CRAT) deficiency has previously been shown to result in muscle insulin resistance due to accumulation of long-chain acylcarnitines. However, differences in the acylcarnitine profile and/or changes in gene expression and protein abundance of CRAT in myotubes obtained from obese patients with type 2 diabetes mellitus (T2DM) and glucose-tolerant obese and lean controls remain unclear. The objective of the study was to examine whether myotubes from obese patients with T2DM express differences in gene expression and protein abundance of CRAT and in acylcarnitine species pre-cultured under glucose and insulin concentrations similar to those observed in healthy individuals in the over-night fasted, resting state. Primary myotubes obtained from obese persons with or without T2DM and lean controls (n=9 in each group) were cultivated and harvested for LC-MS-based profiling of acylcarnitines. The mRNA expression and protein abundance of CRAT were determined by qPCR and Western Blotting, respectively. Our results suggest that the mRNA levels and protein abundance of CRAT were similar between groups. Of the 14 different acylcarnitine species measured by LC-MS, the levels of palmitoylcarnitine (C16) and octadecanoylcarnitine (C18) were slightly reduced in myotubes derived from T2DM patients (p<0.05) compared to glucose-tolerant obese and lean controls. This suggests that the CRAT function is not the major contributor to primary insulin resistance in cultured myotubes obtained from obese T2DM patients.

| S-EPMC5614545 | biostudies-literature

A brain-derived metric for preferred kinetic stimuli.

Project description:We here address the question of whether there is any correlation between subjective preference for simple configurations within a specific visual domain such as motion and strength of activity in visual areas in which that domain is emphasized. We prepared several distinctive patterns of dots in motion with various characteristics and asked humans to rate them according to their preference, before and while scanning the activity in their brains with functional magnetic resonance imaging. For simplicity, we restricted ourselves to motion in the fronto-parallel plane. Moving patterns produced activity in areas V1, V2, the V3 complex (V3, V3A, V3B) and V5, but only in areas V5, V3A/B and parietal cortex did the preferred kinetic patterns produce stronger activity when compared with the non-preferred ones. In addition, preferred patterns produced activity within field A1 of medial orbito-frontal cortex (mOFC), which is not otherwise activated by kinetic stimuli. Hence, for these areas, stronger neural activity correlated with subjective preference. We conclude that configurations of kinetic stimuli that are subjectively preferred correlate with stronger activity within early visual areas and within mOFC. This opens up the possibility of more detailed studies to relate subjective preferences to strength of activity in early visual areas and to relate activity in them to areas whose activity correlates with the subjective experience of beauty.

| S-EPMC3352092 | biostudies-literature

pH-dependence of carnitine acetyltransferase activity.

Project description:1. The pH-dependence of the kinetic constants of the carnitine acetyltransferase reaction has been investigated with the enzyme from pigeon breast muscle. 2. Michaelis constants for (-)-carnitine and acetyl-(-)-carnitine vary in a similar fashion in the pH range 6.0-9.0. A single ionizing group on the enzyme with an apparent pK7.2 is required in the basic form for binding of these substrates. 3. Binding of CoASH or acetyl-CoA raises the apparent pK of an ionizing group on the enzyme from 7.85 to 8.25. This group is probably not directly involved in forming the enzyme-substrate complex, but its microscopic environment is presumably altered. Another group in either the substrate or the free enzyme, with an apparent pK6.4, is needed in the basic form for optimum binding of CoA substrates. 4. This last group has been unequivocally identified as the 3'-phosphate of CoA, by showing that the K(m) of carnitine acetyltransferase for the substrate acetyl-3'-dephospho-CoA is independent of pH in the range 6.0-7.8. 5. V'(max.), the maximum velocity of the catalysed reaction between acetyl-CoA and (-)-carnitine, is constant between pH6.0 and 8.8. 6. The significance of these results in terms of a previously postulated reaction scheme for this enzyme is discussed.

| S-EPMC1270612 | biostudies-other

The substrate specificity of carnitine acetyltransferase.

Project description:1. A study of the acyl group specificity of the carnitine acetyltransferase reaction [acyl-(-)carnitine+CoASH right harpoon over left harpoon (-)-carnitine+acyl-CoA] has been made with the enzyme from pigeon breast muscle. Acyl groups containing up to 10 carbon atoms are transferred and detailed kinetic investigations with a range of acyl-CoA and acylcarnitine substrates are reported. 2. Acyl-CoA derivatives with 12 or more carbon atoms in the acyl group are potent reversible inhibitors of carnitine acetyltransferase, competing with acetyl-CoA. Lauroyl- and myristoyl-CoA show a mixed inhibition with respect to (-)-carnitine, but palmitoyl-CoA competes strictly with this substrate also. Palmitoyl-dl-carnitine shows none of these effects. 3. Ammonium palmitate inhibits the enzyme competitively with respect to (-)-carnitine and non-competitively with respect to acetyl-CoA. 4. It is suggested that a hydrophobic site exists on the carnitine acetyltransferase molecule. The hydrocarbon chain of an acyl-CoA derivative containing eight or more carbon atoms in the acyl group may interact with this, which results in enhanced acyl-CoA binding. Competition occurs between ligands bound to this hydrophobic site and the carnitine binding site. 5. The possible physiological significance of long-chain acyl-CoA inhibition of this enzyme is discussed.

| S-EPMC1270613 | biostudies-other

Cloning and characterization of murine carnitine acetyltransferase: evidence for a requirement during cell cycle progression.

Project description:We have employed a newly developed differential screening technique (reverse strand priming) to identify murine carnitine acetyltransferase (CARAT) as a growth- and cell cycle-regulated gene in S3T3 mouse fibroblasts. Sequence analysis of the full-length cDNA clone and homology comparisons have revealed 87% homology to the human CARAT gene. On Northern blots we were able to measure a 2-3-fold induction 18 h after a mitogenic stimulus following serum deprivation as well as after release from a sodium butyrate block. The cell cycle induction pattern of the CARAT gene was analysed in mouse fibroblasts at different stages of the unperturbed cell cycle. Fractions obtained by elutriation of an exponentially growing culture showed a biphasic maximum of transcript abundance in the G1 and G2 phases of the cell cycle. CARAT expression was investigated in several organs of the adult mouse. Among those measured, CARAT expression was highest, relative to liver, in heart muscle (56-fold) and testis (21-fold). Using both conventional antisense oligodeoxynucleotides and novel single-stranded antisense phagemid DNA, we obtained evidence that the CARAT enzyme function is necessary for progression through G1 and into the S-phase of the cell cycle.

| S-EPMC1218205 | biostudies-other

Muscle-specific deletion of carnitine acetyltransferase compromises glucose tolerance and metabolic flexibility.

Project description:The concept of "metabolic inflexibility" was first introduced to describe the failure of insulin-resistant human subjects to appropriately adjust mitochondrial fuel selection in response to nutritional cues. This phenomenon has since gained increasing recognition as a core component of the metabolic syndrome, but the underlying mechanisms have remained elusive. Here, we identify an essential role for the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT), in regulating substrate switching and glucose tolerance. By converting acetyl-CoA to its membrane permeant acetylcarnitine ester, CrAT regulates mitochondrial and intracellular carbon trafficking. Studies in muscle-specific Crat knockout mice, primary human skeletal myocytes, and human subjects undergoing L-carnitine supplementation support a model wherein CrAT combats nutrient stress, promotes metabolic flexibility, and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility.

| S-EPMC3348515 | biostudies-literature

Crystal structures of murine carnitine acetyltransferase in ternary complexes with its substrates.

Project description:Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine acetyltransferase (CrAT) in a ternary complex with its substrates acetyl-CoA and carnitine, and the structure of the S554A/M564G double mutant in a ternary complex with the substrates CoA and hexanoylcarnitine. Detailed analyses suggest that these structures may be good mimics for the Michaelis complexes for the forward and reverse reactions of the enzyme, representing the first time that such complexes of CrAT have been studied in molecular detail. The structural information provides significant new insights into the catalytic mechanism of CrAT and possibly carnitine acyltransferases in general.

| S-EPMC2940834 | biostudies-literature

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