scholarly journals Active sites residues of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT-II)

1998 ◽  
Vol 330 (2) ◽  
pp. 1029-1036 ◽  
Author(s):  
Nóirin NIC a'BHÁIRD ◽  
Victoria YANKOVSKAYA ◽  
R. Rona RAMSAY
Keyword(s):  
Active Sites ◽  
Chemical Reactivity ◽  
Fatty Acyl ◽  
Carnitine Palmitoyltransferase ◽  
Inactivation Kinetics ◽  
Beef Liver ◽  
First Order Kinetics ◽  
Carnitine Acyltransferases ◽  
Carnitine Palmitoyltransferase Ii ◽  
Selective Reagent

The carnitine acyltransferases which catalyse the reversible transfer of fatty acyl groups between carnitine and coenzyme A have been proposed to contain a catalytic histidine. Here, the chemical reactivity of active site groups has been used to demonstrate differences between the active sites of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase-II (CPT-II). Treatment of CPT-II with the histidine-selective reagent, diethyl pyrocarbonate (DEPC), resulted in simple linear pseudo-first-order kinetics. The reversal of the inhibition by hydroxylamine and the pKa (7.1) of the modified residue indicated that the residue was a histidine. The order of the inactivation kinetics showed that 1 mol of histidine was modified per mol of CPT-II.

Increased muscle carnitine palmitoyltransferase II mRNA after increased contractile activity

1995 ◽  
Vol 268 (2) ◽  
pp. E277-E281 ◽  
Author(s):  
Z. Yan ◽  
S. Salmons ◽  
J. Jarvis ◽  
F. W. Booth
Keyword(s):  
Skeletal Muscle ◽  
Fatty Acids ◽  
Contractile Activity ◽  
Fatty Acyl ◽  
Carnitine Palmitoyltransferase ◽  
The Matrix ◽  
Wet Weight ◽  
Matrix Compartment ◽  
Dependent Transport ◽  
Carnitine Palmitoyltransferase Ii

The capacity of skeletal muscle to oxidize fatty acids increases with endurance training. The oxidation of long-chain fatty acids occurs in mitochondria and is initiated by a carnitine-dependent transport step in which three enzymes help fatty acyl groups enter the matrix compartment. The purpose of this study was to determine whether pretranslational regulation of one of these three enzymes, carnitine palmitoyltransferase II (CPT II), as estimated from the level of CPT II mRNA, plays a role in the doubling of CPT activity in skeletal muscle of rats subjected to daily 2-h bouts of running on treadmills (P. A. Mole, L. B. Oscai, and J. O. Holloszy. J. Clin. Invest. 50: 2323-2330, 1971). After 100 min/day of running on motor-driven treadmills for 2 wk, CPT II mRNA in the plantaris muscle was unchanged when normalized per unit of extracted RNA but was 50% higher (P < 0.05) over sedentary controls when normalized per unit of muscle wet weight. To test whether additional contractile activity would make CPT II mRNA even higher, continuous indirect electrical stimulation was imposed on the tibialis anterior muscles. After 9 days of chronic stimulation, CPT II mRNA was 63, 221, and 137% greater than control (P < 0.001) when normalized to extracted RNA, muscle wet weight, and whole muscle, respectively, compared with the muscle in the control rats. These data indicate that pretranslational regulation of CPT II occurs in response to increased contractile activity in skeletal muscle.

The Oxidation of Fatty-Acyl Derivatives by Mitochondria from Bovine Fetal and Calf Hearts

1971 ◽  
Vol 49 (12) ◽  
pp. 1296-1300 ◽  
Author(s):  
J. T. Brosnan ◽  
I. B. Fritz
Keyword(s):  
Ionic Strength ◽  
Fatty Acyl ◽  
Carnitine Palmitoyltransferase ◽  
Basal Rate ◽  
Rate Of Oxygen Consumption ◽  
Carnitine Acyltransferases ◽  
Substrate Respiration ◽  
Fetal Bovine ◽  
Carnitine Palmitoyltransferase Activity ◽  
Acyl Derivatives

Functional activity of the "external" carnitine palmitoyltransferase in intact mitochondria, prepared from hearts from various sources, was estimated by measuring respiration by mitochondria in the presence of palmitoyl-CoA plus or minus l-carnitine. When palmitoyl-CoA alone was substrate, respiration was not increased above the basal rate under all conditions examined. Addition of l-carnitine increased respiration, provided the ionic strength of the incubation medium was sufficiently high. In the presence of palmitoyl-CoA plus l-carnitine, the rate of respiration increased as the ionic strength was increased to physiological levels. In contrast, the increase in rate of oxygen consumption by heart mitochondria which followed the addition of palmitoyl-l-carnitine was relatively independent of the ionic strength of the medium.Mitochondrial fractions prepared from fetal bovine hearts were shown to possess "external" carnitine palmitoyltransferase activity, as judged by the ability of l-carnitine to stimulate respiration by mitochondria incubated with palmitoyl-CoA under various conditions. These data were discussed in relation to information available concerning the functions of different carnitine acyltransferases in mitochondria.

Properties of a Purified Carnitine Palmitoyltransferase, and Evidence for the Existence of Other Carnitine Acyltransferases

1971 ◽  
Vol 49 (8) ◽  
pp. 941-948 ◽  
Author(s):  
Bozena Kopec ◽  
Irving B. Fritz
Keyword(s):  
Chain Length ◽  
Acyl Chain ◽  
High Specificity ◽  
Apparent Molecular Weight ◽  
Relative Activity ◽  
Fatty Acyl ◽  
Carnitine Palmitoyltransferase ◽  
Fatty Acyl Chain ◽  
Acyl Chain Length ◽  
Carnitine Palmitoyltransferase Ii

Carnitine palmitoyltransferase (CPT) has been purified, and some of its properties are described. The purified preparations had specific activities of 10–38 μmoles/min/mg protein at 35°. The apparent molecular weight of CPT was approximately 75 000, or a multiple thereof. The K′m value for palmitoyl-CoA was 1.8 × 10−5 M, while the K′s values for (–)-carnitine, CoA–SH, and (–)-palmitoylcarnitine were estimated to be 4.5 × 10−4 M, 5.5 × 10−6 M, and 1.4 × 10−4 M, respectively. The enzyme was subject to inhibition by all substrates and products. CPT had a relatively broad fatty acyl chain-length specificity, with highest Vmax values obtained for the transfer of myristoyl and palmitoyl groups. K′m values were lowest with long-chain fatty acylcarnitinè derivatives (1.9 × 10−4 M for (–)-stearoylcarnitine versus 1.2 × 10−3 M for (–)-decanoylcarnitine).Evidence is presented demonstrating the existence of two additional protein fractions possessing carnitine acyltransferase activities with different fatty acyl chain-length specificities. One, which displayed highest relative activity towards the transfer of octanoyl groups, is tentatively designated carnitine octanoyltransferase. The other, which displayed a high specificity toward the transfer of palmitoyl and stearoyl groups from acylcarnitine when preincubated with CoA–SH, is tentatively designated carnitine palmitoyltransferase II.

scholarly journals Expression of precursor and mature carnitine palmitoyltransferase II in Escherichia coli and in vitro: differential behaviour of rat and human isoforms

1993 ◽  
Vol 294 (1) ◽  
pp. 79-86 ◽  
Author(s):  
N F Brown ◽  
A Sen ◽  
D A Soltis ◽  
B Jones ◽  
D W Foster ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Sites ◽  
Expression System ◽  
Active Form ◽  
Carnitine Palmitoyltransferase ◽  
Coding Region ◽  
E Coli ◽  
Catalytically Active ◽  
Carnitine Palmitoyltransferase Ii

cDNAs corresponding to the precursor and mature forms of rat carnitine palmitoyltransferase II (CPT II) were found to be readily expressed in Escherichia coli. In both cases, catalytically active immunoreactive protein was produced and became largely membrane-associated. The precursor form of the enzyme was not proteolytically processed. Removal of 126 bp from the 5′ end of the cDNA coding region allowed expression of a truncated CPT II (lacking the N-terminal 17 residues of the mature protein), but this product was inactive. cDNAs encoding the precursor and mature forms of human CPT II resisted direct expression in E. coli. However, the impediment was overcome when the latter cDNA was ligated in-frame 3′ to sequence encoding a glutathione S-transferase. This construct yielded abundant quantities of the corresponding fusion protein, a portion of which was soluble and catalytically active. In vitro transcription and translation of the various cDNAs established that the lower mobility on SDS/PAGE of rat CPT II compared with its human counterpart (despite their identical numbers of amino acids) is an intrinsic property of the primary sequences of the proteins themselves. Also, the human cDNA was found to contain an artifactual termination signal for T3 RNA polymerase that could be bypassed by the T7 polymerase. Thus rat CPT II can be expressed in active form in E. coli with characteristics similar to those of the enzyme in mitochondria, opening the way to future location of active sites within the molecule. An alternative expression system will be needed for similar studies on human CPT II.

scholarly journals Density Functional Theory and Molecular Docking Investigations of the Chemical and Antibacterial Activities for 1-(4-Hydroxyphenyl)-3-phenylprop-2-en-1-one

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3631
Author(s):  
Ahmed M. Deghady ◽  
Rageh K. Hussein ◽  
Abdulrahman G. Alhamzani ◽  
Abeer Mera
Keyword(s):  
Density Functional Theory ◽  
Antibacterial Activity ◽  
Molecular Docking ◽  
Carbonyl Group ◽  
Active Sites ◽  
Density Functional ◽  
Chemical Reactivity ◽  
Basis Set ◽  
Molecular Docking Study ◽  
Functional Theory

The present investigation informs a descriptive study of 1-(4-Hydroxyphenyl) -3-phenylprop-2-en-1-one compound, by using density functional theory at B3LYP method with 6-311G** basis set. The oxygen atoms and π-system revealed a high chemical reactivity for the title compound as electron donor spots and active sites for an electrophilic attack. Quantum chemical parameters such as hardness (η), softness (S), electronegativity (χ), and electrophilicity (ω) were yielded as descriptors for the molecule’s chemical behavior. The optimized molecular structure was obtained, and the experimental data were matched with geometrical analysis values describing the molecule’s stable structure. The computed FT-IR and Raman vibrational frequencies were in good agreement with those observed experimentally. In a molecular docking study, the inhibitory potential of the studied molecule was evaluated against the penicillin-binding proteins of Staphylococcus aureus bacteria. The carbonyl group in the molecule was shown to play a significant role in antibacterial activity, four bonds were formed by the carbonyl group with the key protein of the bacteria (three favorable hydrogen bonds plus one van der Waals bond) out of six interactions. The strong antibacterial activity was also indicated by the calculated high binding energy (−7.40 kcal/mol).

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