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P C Engel

Publications and source records attributed to P C Engel.

At least 91 records · Page 5Linked to original sources
PubMed1991

Heterogeneity for mutations in medium chain acyl-CoA dehydrogenase deficiency in the UK population.

MCAD is the commonest inherited disorder of fatty acid oxidation. We have sought for and studied 21 affected children from 18 families within the UK. In 14 families the children are homozygous for the G985 mutation. In three families the children are compound heterozygotes for G985 and thus carry another and unknown mutation. In one family the child does not carry the G985 mutation on either allele. The carrier incidence of the G985 mutation is 1 in 68, which suggests that the natural history of MCAD deficiency deserves further study.

Acyl-CoA Dehydrogenases↗
PubMed1990

A pH-dependent activation-inactivation equilibrium in glutamate dehydrogenase of Clostridium symbiosum.

1. On transferring Clostridium symbiosum glutamate dehydrogenase from pH 7 to assay mixtures at pH 8.8, reaction time courses showed a marked deceleration that was not attributable to the approach to equilibrium of the catalysed reaction. The rate became approximately constant after declining to 4-5% of the initial value. Enzyme, stored at pH 8.8 and assayed in the same mixture, gave an accelerating time course with the same final linear rate. The enzyme appears to be reversibly converted from a high-activity form at low pH to a low-activity form at high pH. 2. Re-activation at 31 degrees C upon dilution from pH 8.8 to pH 7 was followed by periodic assay of the diluted enzyme solution. At low ionic strength (5 mM-Tris/HCl), no re-activation occurred, but various salts promoted re-activation to a limiting rate, with full re-activation in 40 min. 3. Re-activation was very temperature-dependent and extremely slow at 4 degrees C, suggesting a large activation energy. 4. 2-Oxoglutarate, glutarate or succinate (10 mM) accelerated re-activation; L-glutamate and L-aspartate were much less effective. 5. The monocarboxylic amino acids alanine and norvaline appear to stabilize the inactive enzyme: 60 mM-alanine does not promote re-activation, and, as substrates at pH 8.8 for enzyme stored at pH 7, alanine and norvaline give progress curves showing rapid complete inactivation. 6. Mono- and di-nucleotides (AMP, ADP, ATP, NAD+, NADH, NADP+, CoA, acetyl-CoA) at low concentrations (10(-4)-10(-3) M) enhance re-activation at pH 7 and also retard inactivation at pH 8.8. 7. The re-activation rate is independent of enzyme concentration: ultracentrifuge experiments show no changes in molecular mass with or without substrates. 8. The activation-inactivation appears to be due to a slow pH-dependent conformational change that is sensitively responsive to the reactants and their analogues.

Adenine Nucleotides↗
PubMed1990

Octanoylation of the lipoyl domains of the pyruvate dehydrogenase complex in a lipoyl-deficient strain of Escherichia coli.

The overexpression of a subgene encoding a hybrid lipoyl domain of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli has previously been shown to result in the formation of lipoylated and unlipoylated products. Overexpression of the same subgene in a lipoic acid biosynthesis mutant growing under lipoate-deficient conditions has now been shown to produce domains modified by octanoylation as well as unmodified domains. It was concluded from the mass of a lipoyl-binding-site peptide that the modification involves N6-octanoylation of the lysine residue (Lys244) that is normally lipoylated, and this was confirmed by the trypsin-insensitivity of the corresponding Lys244-Ala-245 bond, and the absence of modification in a mutant domain in which Lys244 is replaced by Gln. This novel protein modification raises interesting questions concerning the pathway of lipoic acid biosynthesis and the mechanism of enzyme lipoylation.

Amino Acid Sequence↗
PubMed1988

Acylcoenzyme A dehydrogenase deficiency in heart tissue from infants who died unexpectedly with fatty change in the liver.

Heart muscle from infants who died unexpectedly and who showed fatty changes in the liver at necropsy was analysed for long chain and medium chain acylcoenzyme A dehydrogenase activities by using the natural electron acceptor. In two of the seven cases investigated a deficiency in acylcoenzyme A dehydrogenase activity was found. In one case the deficiency was in medium chain acylcoenzyme A dehydrogenase activity and in the other long chain acylcoenzyme A dehydrogenase activity. These findings emphasise the importance of investigating fatty acid oxidation in infants who have died unexpectedly.

Acyl-CoA Dehydrogenase↗
PubMed1987

CoA-persulphide: a possible in vivo inhibitor of mammalian short-chain acyl-CoA dehydrogenase.

The characteristic green colour of native short-chain acyl-CoA dehydrogenases (EC 1.3.99.2) results from a charge transfer complex between the FAD prosthetic group and a tightly bound molecule of CoA-persulphide. The native enzyme from ox liver mitochondria was found to have about 60% of its FAD cofactor liganded with CoA-persulphide. When artificially fully liganded with CoA-persulphide, this enzyme was inhibited by 90% in comparison to unliganded enzyme. Enzymic activity could be slowly restored by displacing the CoA-persulphide with high concentrations of butyryl-CoA, the enzyme's physiological substrate. The results show that CoA-persulphide is a potent inhibitor of short-chain acyl-CoA dehydrogenase and may have a physiological role in the regulation of beta-oxidation.

Acyl-CoA Dehydrogenase↗
PubMed1985

The suicide inactivation of ox liver short-chain acyl-CoA dehydrogenase by propionyl-CoA. Formation of an FAD adduct.

Propionyl-CoA gave an unexpectedly low turnover (0.03 s-1) and high Km (153 microM) as a substrate for ox liver short-chain acyl-CoA dehydrogenase (SCAD). On addition of an excess of propionyl-CoA to SCAD the flavin A448 decreased to about 30% of its original value and the peak at 368 nm was replaced by one at 335 nm. The decrease in A448 exhibited first-order kinetics and correlated with a first-order decrease in the enzyme's catalytic activity to 22% of the initial value. The flavin, released from propionyl-CoA-treated enzyme with trichloroacetic acid, reacted with O2 to form a stable free radical. This suggests that a reduced N-5 flavin adduct is formed on the enzyme and protected from O2. The released adduct was separated from unmodified flavin and excess propionyl-CoA by h.p.l.c., and was shown by 3H-labelling to contain CoA. The incompleteness of the decrease in the enzyme's A448 and specific activity on incubation with propionyl-CoA probably reflects an equilibrium between covalently and non-covalently bound acyl-CoA, since the spectral changes could be reversed. The enzyme was also re-activated by dilution and incubation with a large molar excess of butyryl-CoA. The rate constant, approx. 2 X 10(-3) s-1, for re-activation, taken with the extrapolated rate constant for the opposing inactivation reaction, 8.9 X 10(-3) s-1, explains the 22% residual activity at equilibrium. The results suggest that propionyl-CoA is a suicide inhibitor for SCAD.

Acyl Coenzyme A↗
PubMed1985

Defects of metabolism of fatty acids in the sudden infant death syndrome.

Two hundred consecutive cases of the sudden infant death syndrome were reviewed for the presence of fat in the liver; 14 showed diffuse panlobular microvesicular fatty change indistinguishable from that found in Reye's syndrome. Samples of frozen liver were available in five of the 14 cases; histochemical analysis showed well preserved cytochrome oxidase and succinate dehydrogenase activity in all five, uncharacteristic of Reye's syndrome. Fatty acyl-coenzyme A dehydrogenase activity in the liver was assayed biochemically in two of the same five cases with severe hepatic fatty infiltration; both showed a defect in medium chain acyl-coenzyme A dehydrogenase activity using the substrate octanoyl-coenzyme A. Both cases also showed cerebral oedema in association with fatty infiltration of renal tubules, myocardium, and skeletal muscle, characteristic of Reye's syndrome. It is concluded that diffuse panlobular microvesicular fatty change of the liver in victims of the sudden infant death syndrome, although essentially non-specific, indicates that the state of mitochondrial enzymes should be investigated.

Acyl-CoA Dehydrogenase↗
PubMed1985

Crystallization of an NAD+-dependent glutamate dehydrogenase from Clostridium symbiosum.

Crystals of a bacterial NAD+-dependent glutamate dehydrogenase (GDHase) have been grown over a wide range of pH values by using the hanging drop method of vapour diffusion with ammonium sulphate as the precipitant. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of this enzyme together with high pressure liquid chromatography/gel filtration, shows that this GDHase is hexameric like the GDHases of vertebrates. X-ray photographs of the crystals show that they diffract to at least 2.0 A, and an analysis of the diffraction pattern demonstrates that the hexamer is arranged in at least pseudo 32 symmetry.

Chromatography, Gel↗
PubMed1985

Kinetic studies of ox-liver glutamate dehydrogenase oxidative deamination of two glutamate analogues, L-threo-gamma-methylglutamate and L-alpha-amino-gamma-nitraminobutyrate, in the presence of the allosteric effector ADP.

Ox-liver glutamate dehydrogenase is known to utilise a wide range of amino acid substrates. Kinetic studies are presented here for L-threo-gamma-methylglutamate and L-alpha-amino-gamma-nitraminobutyrate in the presence of the allosteric effector ADP. The results presented are considered in the light of similar studies presented elsewhere in which the cofactor was systematically replaced by a variety of analogues. These amino acid analogues share the same pH optimum as glutamate, unlike the monocarboxylic amino acids including alanine and norvaline, and give linear double-reciprocal plots under the conditions used here. Studies with the alternative coenzymes have suggested an ordered addition of glutamate before coenzyme in the presence of ADP. The present results obtained under identical conditions support this conclusion.

Adenosine Diphosphate↗
PubMed1984

The kinetic mechanism of ox liver glutamate dehydrogenase in the presence of the allosteric effector ADP. The oxidative deamination of L-glutamate.

In steady-state kinetic studies of ox liver glutamate dehydrogenase in 0.11 M-potassium phosphate buffer, pH7, at 25 degrees C, the concentration of ADP was varied from 0.5 to 1000 microM. Inhibition was observed except when the concentrations of both glutamate and coenzyme were high, when activation was seen. With NAD+ or NADP+ as coenzyme, 200 microM-ADP was sufficient to saturate the enzyme with respect to the major effect of this nucleotide. In the presence of 210 microM-ADP, widely varied concentrations of coenzyme give linear Lineweaver-Burk plots, in marked contrast with results obtained previously for kinetics without ADP. This has allowed evaluation for the reaction with NAD+, NADP+ and acetylpyridine-adenine dinucleotide (315 microM-ADP in the last case) of all four initial rate parameters, i.e. the phi coefficients in the equation: (Formula: see text) where A is coenzyme and B is glutamate. The relative constancy of phi B and of phi AB/phi A with the different coenzymes point to a compulsory-order mechanism with glutamate as the leading substrate. This conclusion, though unexpected, agrees well with various previous observations on the binding of oxidized coenzyme.

Adenosine Diphosphate↗
PubMed1984

A kinetic study of the oxidative deamination of L-glutamate by Peptostreptococcus asaccharolyticus glutamate dehydrogenase using a variety of coenzymes.

The NAD+-specific glutamate dehydrogenase from Peptostreptococcus asaccharolyticus follows Michaelis-Menten kinetics in contrast to the enzyme from several other sources, and thus gives linear double-reciprocal plots of initial-rate data. The initial-rate parameters have been determined for this bacterial dehyrogenase in the direction of oxidative deamination. The use of alternative coenzymes leads to some conclusions about the order of substrate addition. An investigation of the pH dependence of this reaction reveals that the binding of oxidised coenzyme is independent of pH over the range 6-9. The kinetic data are consistent with an ordered addition of coenzyme prior to glutamate, the reverse of the mechanism derived with ox glutamate dehydrogenase in the presence of ADP.

Coenzymes↗
PubMed1984

Ox liver glutamate dehydrogenase. The use of chemical modification to study the relationship between catalytic sites for different amino acid substrates and the question of kinetic non-equivalence of the subunits.

The effect of pyridoxal 5'-phosphate on the activity of ox liver glutamate dehydrogenase towards different amino acid substrates was investigated. Both alanine and glutamate activities decreased steadily in the presence of pyridoxal 5'-phosphate. The alanine/glutamate activity ratio increased as a function of inactivation by pyridoxal 5'-phosphate, indicating that glutamate activity is lost more rapidly than alanine activity. A mixture of NADH, GTP and 2-oxoglutarate completely protected the alanine and glutamate activities against inactivation by pyridoxal 5'-phosphate. The activity of glutamate dehydrogenase towards glutamate and leucine decreased steadily in a constant ratio in the presence of pyridoxal 5'-phosphate. The effect of leucine on the alanine and glutamate activities as a function of inactivation by pyridoxal 5'-phosphate was studied. The results are interpreted to suggest that the subunits of glutamate dehydrogenase hexamer are kinetically non-equivalent with regard to activity towards the two monocarboxylic amino acids as well as glutamate, and that all three substrates share the same active centre. However, leucine is also able to bind at a separate regulatory site.

Alanine↗
PubMed1984

The purification and properties of ox liver short-chain acyl-CoA dehydrogenase.

The FAD-containing short-chain acyl-CoA dehydrogenase was purified from ox liver mitochondria by using (NH4)2SO4 fractionation, DEAE-Sephadex A-50 and chromatofocusing on PBE 94 resin. The enzyme is a tetramer, with a native Mr of approx. 162 000 and a subunit Mr of 41 000. Short-chain acyl-CoA dehydrogenases are usually isolated in a green form. The chromatofocusing step in the purification presented here partially resolved the enzyme into a green form and a yellow form. In the dye-mediated assay system, the enzyme exhibited optimal activity towards 50 microM-butyryl-CoA at pH 7.1. Kinetic parameters were also determined for a number of other straight-chain acyl-CoA substrates. The u.v.- and visible-absorption characteristics of the native forms of the enzyme are described, together with complexes formed by addition of butyryl-CoA, acetoacetyl-CoA and CoA persulphide.

Acetyl Coenzyme A↗
PubMed1984

Butyryl-CoA dehydrogenase from Megasphaera elsdenii. Specificity of the catalytic reaction.

The absorption coefficient of butyryl-CoA dehydrogenase from Megasphaera elsdenii at 450 nm is determined as 14.4 mM-1 X cm-1 in the CoA-free form and 14.2 mM-1 X cm-1 in the CoA-liganded form (both yellow). The latter value is considerably higher than the earlier published estimate. Phenazine ethosulphate offers great advantages over phenazine methosulphate as a coupling dye in the catalytic assay despite giving lower Vmax. values (506 min-1 as compared with 1250 min-1 under the conditions used). The phenazine ethosulphate assay is used to establish a pH optimum of 8.05 for oxidation of 100 microM-butyryl-CoA. The rates of oxidation of a range of straight-chain, branched-chain and alicyclic acyl thioesters are used to provide the following information. Only straight-chain acyl groups containing 4-6 carbon atoms are easily accommodated by the postulated hydrophobic pocket of the enzyme. C-3-substituted acyl-CoA thioesters are not oxidized at a significant rate, suggesting that the C-3 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Acyl-CoA thioesters with substitutions at C-2 are oxidized, though at a lower rate than their straight-chain counterparts. This implies that the C-2 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Saturated alicyclic carboxylic acyl-CoA thioesters with 4-7 carbon atoms in the ring are oxidized, with maximal activity for the cyclohexane derivative. This implies that optimal oxidation requires a true trans orientation of the two departing hydrogen atoms. The strain imposed by bound unsaturated alicyclic acyl thioesters strikingly perturbs the flavin visible-absorption spectrum, with the exception of the cyclohex-2-ene derivative, which forms a complex with similar spectral properties to those of the crotonyl-CoA complex. In the thiol moiety of thioester substrates the amide bond of N-acetylcysteamine is essential for both binding and catalysis. The adenosine structure contributes substantially to strong binding, but is less important in determining the catalytic rate.

Acyl Coenzyme A↗
PubMed1984

Glutaric aciduria type II: biochemical investigation and treatment of a child diagnosed prenatally.

Two sibs with the acute neonatal form of glutaric aciduria type II (deficient in vivo activity of multiple acyl-CoA dehydrogenases) are described. In the second case diagnosis was made prenatally on the basis of reduced oxidation of palmitate by cultured amniotic fluid cells. With prompt intervention in the neonatal period and a carefully controlled diet later, this second progressed well up to 4 months of age but died suddenly of cardiac failure, probably attributable to accumulation of fat. Neither patient showed any congenital morphological abnormality. Cultured fibroblasts from the second case showed a marked defect in the oxidation of a range of substrates requiring acyl-CoA dehydrogenases for their catabolism, but residual activity for some substrates was quite high. Large quantities of sarcosine were excreted in urine, again suggesting that the mutation leaves some residual dehydrogenation activity. Butyryl-, octanoyl- and palmitoyl-CoA dehydrogenases were present in essentially normal quantities in postmortem liver.

Adipates↗
PubMed1984

Characterization of Peptostreptococcus asaccharolyticus glutamate dehydrogenase purified by dye-ligand chromatography.

Glutamate dehydrogenase (L-glutamate:NAD+ oxidoreductase (deaminating); EC 1.4.1.2) has been purified from Peptostreptococcus asaccharolyticus in a single step using dye-ligand chromatography. The enzyme (GDH) was present in high yields and was stabilized in crude extracts. A subunit molecular weight of 49000 +/- 500 was determined by SDS polyacrylamide gel electrophoresis and six bands were obtained after cross-linking the subunits with dimethyl suberimidate. This bacterial GDH was predominantly NAD+-linked, but was able to utilize both NADP+ and NADPH at 4% of the rates with NAD+ and NADH, respectively. An investigation of the amino acid specificity revealed some similarities with GDH from mammalian sources and some clear differences. The values of apparent Km for the substrates ammonia, 2-oxoglutarate, NADH, NAD+ and glutamate were 18.4, 0.82, 0.066, 0.031 and 6 mM, respectively. The P. asaccharolyticus GDH was not regulated by purine nucleotides, but was subject to strong inhibition with increasing ionic strength.

Chromatography, Agarose↗