We also conducted viscosity-based stopped-flow experiments that support product release as being rate limiting for the reaction. When this order is reversed, catalysis is decrease by about 100-fold. This preferred pathway involves the ordered binding of substrates, with carnitine binding before acetyl-CoA in the forward direction and acetylcarnitine followed by CoA in the reverse direction. Global analysis of full progress curve data with fluorescence intensity equilibrium titrations of substrates and double mixing experiments have revealed a preferred kinetic pathway in the mammalian CrAT reaction. Thus, we have collected numerous CrAT forward and reverse full progress curves using a stopped-flow rapid mixing device utilizing various mixing strategies directly following the acetyl-CoA to CoA transition in the UV spectrum. Our efforts in global fitting CrAT literature data have revealed deficiencies that can only be circumvented by producing a new collection of CrAT kinetic data. Global analysis overcomes this issue by simultaneously fitting all of the data to the actual model. This method of kinetic analysis, however, does not fit the model itself, but rather predictions of linear patterns. Using Lineweaver-Burk plot analysis, they concluded that CrAT, from pigeon breast muscle, followed a rapid equilibrium bi bi mechanism. To this end, we found early kinetic studies by Chase and Tubbs that provided forward and reverse initial velocity and product inhibition data. Our objective was to generate an accurate kinetic model of mammalian CrAT. Thus, the capacity of CrAT to alleviate metabolic stress via acetyl-CoA/CoA buffering is of special interest to human health. CrAT buffering of acetyl-CoA by the production of acetylcarnitine alleviates metabolic stress by reducing protein acetylation, and by replenishing the CoA pool. When mitochondrial acetyl-CoA levels exceed TCA cycle flux, excess acetyl-CoA may lead to protein acetylation, a ubiquitous metabolic regulator. FAO and GO are the main sources of mitochondrial acetyl-CoA. More broadly, CrAT mediated mitochondrial acetyl-CoA/CoA buffering is related to the balance between glucose oxidation (GO), fatty acid oxidation (FAO), and amino acid oxidation (AAO) – a central topic in metabolism termed fuel selection. CrAT function has also been linked to muscle endurance and has been shown to be a positive metabolic regulator inhibited by lipid stress and obesity. ĬrAT has also been implicated in glucose sensitivity, with CrAT knockout mice demonstrating glucose insensitivity, underscoring the importance of this reaction to healthy metabolic function. Physiologically this scenario is more probable in times of high ATP demand, for example during exercise, where exercising muscle biopsies demonstrate increased acetyl-CoA and acetylcarnitine levels relative to resting muscle. In the mitochondrial matrix, CrAT-catalyzed consumption of acetyl-CoA and production of CoA is thought to be a critical acetyl-CoA/CoA buffer, for instance when the TCA cycle is overwhelmed as an acetyl-CoA sink. CrAT is localized to the mitochondrial matrix or peroxisomes, but recent evidence also suggests a cytosolic CrAT. 2.3.1.7) catalyzes the reversible transfer of an acetyl group between acetyl-CoA and L-carnitine, forming CoA and acetylcarnitine. Mammalian carnitine acetyltransferase (CrAT) (E. Additional viscosity-based kinetic experiments yielded strong evidence that product release is the rate limiting step in the CrAT-catalyzed reaction. Analysis of available CrAT structures support a substrate conformational change between acetyl-CoA/CoA binary versus ternary complexes. 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. 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. Simultaneous mixing of both substrates with CrAT produced relatively fast kinetics that follows an ordered bi bi mechanism. 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. These recent physiological findings imply that CrAT dysfunction, or its catalytic impairment, may lead to disease. 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. Mammalian carnitine acetyltransferase (CrAT) is a mitochondrial enzyme that catalyzes the reversible transfer of an acetyl group from acetyl-CoA to carnitine.
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