Km For Oxaloacetate Research Articles

The Multiple Roles of the Active Site Loop of Watermelon Glyoxysomal Malate Dehydrogenase

All malate dehydrogenases have a flexible loop that starts to close over the active site region of malate dehydrogenase when cofactor binds and locks down completing the active site when the dicarboxylic acid substrate binds. As such the active site loop appears to play a role in both substrate specificity and correct alignment of the substrate to allow hydride transfer and proton abstraction/donation depending on the direction of the reaction. Combined with available crystal structures, detailed analysis of the 1smk.pdb structure shows the loop in a series of microstates from fully open to a closed state induced by the inhibitor citrate binding (figure 1), sequence conservation suggests that the loop region contains a 19 amino acid stretch from P119 to N137 using the watermelon glyoxysomal enzyme as reference. Clustal Omega Analysis of malate dehydrogenases from prokaryotes to mammals shows 5 families of conservation of the loop region (figure 2) allowing a series of hypotheses about structure function relationships of specific residues within the sequence to be made which have been tested by a combination of computational and site directed mutagenesis wetlab approaches. In addition to the two canonical arginines involved in substrate binding, we have explored every other residue in the loop region. Overall, mutations in the first half of the loop had small impact on Specific Activity while mutations in the second half of the loop had larger impacts. R130 mutants to E or S had the most impact while mutation to A or Q had lesser effects. Three other residues in particular had striking effects on Oxaloacetate saturation, G121A, L133S and N137. L133 and N137, at the C terminal base of the loop, were hypothesized to play a role in the repulsive interactions between substrate and cofactor in the active site. Consistent with this hypothesis, creation of an L133S or D137A mutant showed that concomitant with a reduction in specific activity to 2% and 5% wildtype (respectively), Km for Oxaloacetate was dramatically increased while Km for NADH was decreased somewhat. Finally, a series of mutations of M128 (Q,I and A) were explored with varying effects on specific activity (Q>I>A) and significant effects on Km for Oxaloacetate but lesser or no effects on Km for NADH. The impact of M128 mutations in particular were further explored computationally using DeepDDG analysis and effects on stability correlated with impact on kinetic properties. Overall these studies provide a detailed picture of the complex interactions the flexible loop makes that contribute to both substrate binding and catalysis.

Evolution and Adaptation in Malate Dehydrogenases

What is the evolutionary relationship between structure, function and stability is a central concept in the molecular life sciences. Malate Dehydrogenase is an excellent model enzyme to ask questions about this concept since i) it exists throughout biology, playing a central role in energy metabolism and ii) has a number of isoforms including cytosolic, mitochondrial, and in plants, glyoxysomal: overall the organelle forms are thought to have evolved by symbiosis with bacterial forms and are quite different in sequence and properties than the cytosolic forms. Cytosolic forms tend to have higher affinity for NADH while organelle forms have higher affinity for oxaloacetate, broadly consistent with the metabolic roles of the isoforms. Initially, we compared sequence conservation in 22 mammaliam cytoplasmic forms with watermelon glyoxysomal malate dehydrogenase (WMgMDH). Of 40 conserved amino acids in the cytosolic forms, 36 were conserved in WMgMDH. From the structure of WMgMDH, we used HINT and PyMol to examine atomic interactions both within the protein and with substrate and cofactor of the four residues, L159, A221, I250 and K311. We changed each residue to that resembling the cytosolic form to explore which contributed most to cytosolic like properties. We hypothesized that by changing Leucine 159 to a Valine (L159V), the hydrophobic core and cofactor bonding network would be altered. Changing Alanine 221 to Serine (A221S) would create hydrogen bonding potential at this site. Changing Isoleucine 250 to Valine (I250V) would reduce the hydrophobic area and potential impact stability of the protein. Lastly, changing Lysine 311 to Proline (K311P) may affect an interaction with cofactor due to its close proximity. Each mutation was constructed using QuikChange mutagenesis. Sequence confirmed mutants were expressed and purified using NiNTA Affinity Chromatography. Purified proteins were screened using SDS PAGE to assess purity. Specific Activities and Michaelis Constants for NADH and Oxaloacetate were then determined. The K311P and L159V mutants had specific activities similar to wildtype. I250V and A221T had 2 fold and 8 fold lower specific activities, respectively. Within standard errors, the Km for NADH was similar to wildtype for K311P and I250V, while L159V had a two fold lower Km (at a greater than 95% confidence limit). For oxaloacetate binding (as judged by Km for Oxaloacetate), I250V had a significantly higher Km, while L159V had a significantly lower Km (both at greater than 95% confidence limits). The changes produced by the L159 and I250 mutations result in more “cytosolic like” properties, while K311 mutation produced no significant effect. These findings contribute to our understanding of the subtle changes that can contribute to evolution and adaptation of enzyme structure and function.

PURIFICATION AND CHARACTERIZATION OF A MALATE DEHYDROGENASE FROM PHASEOLUS MUNGO

A malate dehydrogenase (MDH) was identified and isolated from the seeds of the mung bean ( Phaseolus mungo ). The procedure entailed extraction, ammonium sulfate precipitation, ion exchange chromatography on CMSephadex and high performance liquid chromatography on POROS HS-20. The purified protein exhibited a molecular mass of 38 kDa in SDS-polyacrylamide gel electrophoresis under both nonreduced and reduced conditions. The pI was 9.7 by isoelectric focusing. The specific activity of the MDH was estimated to be 199 U/mg. The enzyme expressed its optimum activity at pH 7.2, 35C, and showed stable activity below 40C. The Km for oxaloacetate was 112 μ M. The partial N-terminal amino acid sequence data analysis of the first 20 amino acids of the mung bean MDH revealed 95 and 80% homology with two reported MDH from soya bean ( Glycine max ) and potato ( Solanum tuberosum ), respectively.

The Effect of Valine Substitution for Glycine in the Dimer Interface of Citrate Synthase from Thermoplasma acidophilum on Stability and Activity

To determine the role of hydrophobic interactions in the dimer interface of citrate synthase (CS) from Thermoplasma (Tp) acidophilum in thermostabilization, we have used site-directed mutagenesis to replace Gly 196 by Val on the helix L of the subunit interface. Recombinant wild-type and Gly 196 mutant TpCS enzymes were largely identical in terms of substrate specificities (Km for oxaloacetate and acetyl CoA). However, the mutation not only reduced catalytic activity (about 10-fold) (i.e., Vmax, kcat and specific activity) of the TpCS, but also decreased its thermal and chemical stability. Archaeal citrate synthase is active as a dimer, since residues from both monomers participate in the active site. Our results suggest that Gly196 → Val mutation interferes with dimerization, so that improper dimerization or dissociation of the dimer would have a profound affect on the activity as well as the conformational stability of TpCS.

The effect of replacing the conserved active-site residues His-264, Asp-312 and Arg-314 on the binding and catalytic properties of Escherichia coli citrate synthase.

The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate. Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively. The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA. However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions. This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent. The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold. Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E. coli enzyme. The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure. The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding. The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.

A physiological role of Mn2+ in the regulation of cytosolic phosphoenolpyruvate carboxykinase from rat liver is unlikely.

A cytosolic cell-free system prepared from rat liver was used to study the effect of bivalent cations on the activity of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Steady-state concentrations of oxaloacetate in the range 5-50 microM were generated from increasing concentrations of malate+fumarate (10:1); 2 mM ITP and 3 mM Mg2+ were added as cofactors. Micromolar concentrations of Mn2+, Fe2+ and, to a lesser extent, of Zn2+ and Co2+ were shown to stimulate PEPCK activity. Vmax. (mumol/min per g of liver) increased from 0.67 to 1.68 on addition of 5 microM Fe2+ and to 2.34 with 2 microM Mn2+, whereas no significant effect on the Km for oxaloacetate was observed. The apparent K(a) values (total) were 0.62 microM for Mn2+, 1.48 microM for Zn2+, 1.92 microM for Co2+ and 3.37 microM for Fe2+, being 2-8-fold lower than the corresponding published values. Variations of the free Mn2+ concentration were obtained (a) by increasing the Mn2+ concentration (i.e. activation curve) and (b) by simultaneous addition of Mn2+ and increasing concentrations of the chelating agent EGTA (i.e. inactivation curve). Different results were obtained for the activation and inactivation curves. The inactivation curve showed that PEPCK activity was almost unaffected by variations of the free Mn2+ concentration over the range 0.05-0.15 microM. Under comparable experimental conditions, rat liver arginase (another Mn(2+)-dependent enzyme) was completely inactivated. From kinetic evidence, the existence of two distinct molecular forms of cytosolic rat liver PEPCK with different Mn2+ affinities is postulated. Considering the high affinity of PEPCK for Mn2+ and its relative insensitivity to changes in the free Mn2+ concentration, it seems rather unlikely that changes in the free cation concentration play a major role in regulating PEPCK activity in vivo.

Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid

The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp----Asn and Asp----Gly mutations. The Asp----Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563].

Role of threonine 190 in modulating the catalytic function of malate dehydrogenase from a thermophile Thermus flavus

Random mutagenesis of malate dehydrogenase from a thermophilic bacterium, Thermus flavus AT-62, had revealed that a Thr190----Ile replacement near the essential catalytic residue His187 caused marked modulation of the catalytic properties. For further exploration of a role of the residue at this position, this residue was substituted with each of the other amino acids by site-directed mutagenesis. Most of the mutations except for substitution with Ser caused increases in Km for oxaloacetate and increases Ki for oxaloacetate of 2-110 times. Substitution with His or Pro was characterized by the complete loss of substrate inhibition, along with a marked increase in Km for oxaloacetate. Kinetic analyses of the native and altered malate dehydrogenases at various pHs revealed that both Km and Ki for oxaloacetate decreased proportionally to the decrease in pH from 8.40 to 5.75, whereas kcat was nearly constant within the pH range. Apparent shifts of the optimum pH values toward acidity observed with most of the altered malate dehydrogenases were attributed to the increase in Ki, which facilitated the release from the substrate inhibition at a lower pH. Replacement of Thr310, a possible counterpart with which Thr190 forms a hydrogen bond, by Ile caused changes in the catalytic properties similar to those of the Thr190-substituted enzymes. These results suggest that not only the loss of the hydrogen bond between Thr190 and Thr310 but also properties of the residues introduced at position 190 cause modulation of the catalytic properties, probably through dislocation of the loop structure that contains the catalytic residue His187.

Interaction of ligands with pig heart citrate synthase: conformational changes and catalysis.

The fluorescence polarization of 8-hydroxypyrene (1,3,6)trisulfonate (HPT) increases upon interaction with pig heart citrate synthase. Titration of HPT with increasing concentrations of citrate synthase exhibits a hyperbolic saturation behavior, from which the dissociation constant of the enzyme-HPT complex (3.64 +/- 0.3 microM) was determined. The enzyme-HPT interaction is competitively inhibited by oxaloacetate (but not affected by acetyl CoA) with a Ki of 4.3 +/- 1.8 microM. This value is similar to the dissociation constant (Kd = 4.5 +/- 1.6 microM) for the enzyme-oxalocetate complex (determined in the absence of any effector ligand), as well as to the Km for oxaloacetate (3.9 +/- 0.7 microM) in a steady-state citrate synthase catalyzed reaction at a saturating concentration of acetyl CoA. However, the dissociation constant for the citrate synthase-oxaloacetate complex determined by the urea denaturation method is at least 25-fold lower than those determined by the other methods. This suggests an effector role of urea in strengthening the enzyme-oxaloacetate interaction. At low nondenaturing concentrations, urea inhibits the citrate synthase catalyzed reaction in an uncompetitive manner with respect to oxaloacetate, i.e., the Km for oxaloacetate decreases with an increase in urea concentration. This further suggests that urea stabilizes the interaction between citrate synthase and oxaloacetate. The effect of urea is specific for the substrate oxaloacetate, and not for the substrate analogue, HPT, although both these ligands bind citrate synthase with equal affinities, and protect the enzyme against thermal denaturation with equal magnitudes. The results presented herein are discussed in the light of known conformational states of the enzyme.

Crystalline NAD/NADP-dependent malate dehydrogenase; the enzyme from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

Malate dehydrogenase from Sulfolobus acidocaldarius has been purified 240-fold to apparent electrophoretic homogeneity. The enzyme shows a specific activity of 277 U/mg and crystallizes readily. The relative molecular mass of the native enzyme is estimated as 128,500 by ultracentrifugation. After cross-linking a relative molecular mass of 134,000 is found by sodium dodecyl sulfate gel electrophoresis. Malate dehydrogenase from S. acidocaldarius is composed of four subunits of identical size with a relative molecular mass of 34,000. Active-enzyme sedimentation in the analytical ultracentrifuge indicates that the tetramer is the catalytically active species. Kinetic studies in the direction of oxaloacetate reduction showed a Km for NADH of 4.1 microM and a Km for oxaloacetate of 52 microM. Oxaloacetate exhibits substrate inhibition at higher concentrations, L-malate, NAD and NADP were found to be product inhibitors. The enzymatic activity is inhibited by 2-oxoglutarate but not by the adenosine nucleotides AMP, ADP and ATP. Only low activity is detected in the direction of malate oxidation. Malate dehydrogenase from S. acidocaldarius utilizes both NADH and NADPH to reduce oxaloacetate. The enzyme shows A-side stereospecificity for both nicotinamide dinucleotides.

Structure-function relationship of mitochondrial malate dehydrogenase at high dilution and in multicomponent systems.

The structure-function relationship of mitochondrial malate dehydrogenase was investigated at low enzyme concentration, as well as in the presence of polyethylene glycol (PEG 6000) and structure making ions. Previous reports claimed the dimeric enzyme to undergo dissociation in dilute solution, and PEG-induced pairing of dimers in the crystalline state. Sedimentation analysis and gel filtration in 0.1 M sodium phosphate pH 7.6 plus 1 mM EDTA and 1 mM dithioerythritol prove the enzyme to be a stable dimer at c greater than or equal to 0.2 microgram/ml (5 nM). In the presence of 8-20% (w/v) PEG 6000, association of the dimer to tetramers and higher aggregates is observed. At 20% (w/v) polyethylene glycol, ultracentrifugal analysis yields up to 50% tetramers; chemical cross-linking by glutaraldehyde confirms the association in a qualitative way. The enzymatic properties of mMDH (specific activity, Km for oxaloacetate and NADH) in the absence and in the presence of PEG 6000 are indistinguishable. At high polyethylene glycol concentrations (greater than or equal to 20%), the thermal stability of the enzyme is found to be increased. The fluorescence emission, as well as the far-UV and near-UV circular dichroism remain unaffected. Accumulated evidence from equilibrium experiments at low enzyme concentration and reconstitution kinetics (after dissociation at acid pH) proves the active species of mMDH to be the dimer.

A specific translocator for oxaloacetate transport in chloroplasts

Evidence is provided that oxaloacetate is taken up by chloroplasts from maize and spinach leaves by a high‐affinity carrier. Apparent K m values for oxaloacetate are about 45 and 9 μM, respectively. Uptake was measured by following [4‐14C]oxaloacetate reduction in illuminated chloroplasts. With maize chloroplasts the rates of uptake were sufficient to account for photosynthesis. This carrier had a very low affinity for malate and 2‐oxoglutarate. Ratios of the K i, for malate or 2‐oxoglutarate to the K m for oxaloacetate were about 150 and 50, respectively.

Control of ketogenesis and fatty-acid synthesis at the mitochondrial branch-point for acetyl-CoA in the chick liver cell: effect of adenosine 3',5'-monophosphate.

Previous work has shown that dibutyryl adenosine 3′,5′‐monophosphate (Bt2cAMP) markedly activates ketogenesis from fatty acids released during the mobilization of cytoplasmic triacylglycerol‐rich vesicles in the chick liver cell. In the present investigation, activation of the formation of [14C]acetoacetate and 3‐[14C]hydroxybutyrate from [1‐14C]oleate by the cyclic nucleotide was shown to occur at two mitochondrial sites: (a) the l‐(–)‐carnitine‐dependent translocation of the fatty acid across the inner mitochondrial membrane and (b) the intramitochondrial metabolism of acetyl‐CoA.The hypothesis was tested that cAMP regulates ketogenesis and fatty acid synthesis by controlling acetyl‐CoA flux at the mitochondrial branch‐point leading into the 3‐hydroxy‐3‐methylglutaryl‐CoA cycle or to citrate formation. Bt2cAMP caused an immediate activation of acetoacetate formation and a concomitant inhibition of fatty acid synthesis from acetate. Evidence is presented that these effects are due to a cyclic‐nucleotide‐induced fall in the mitochondrial concentration of oxaloacetate. An assay for oxaloacetate, sufficiently sensitive to analyze extracts of cells in monolayer culture, was employed. With this assay, Bt, cAMP was shown to cause a > 90% decrease in cellular oxaloacetate concentration. The calculated level to which intramitochondrial oxaloacetate fell was below the Km for oxaloacetate in the citrate‐synthase‐catalyzed reaction and, therefore, was sufficient to curtail citrate formation.Lactate, a precursor of oxaloacetate and an antiketogenic agent, reversed the effects of Bt2cAMP. Under conditions where glucose was sole precursor of oxaloacetate and citrate, the activation of ketogenesis and the inhibition of fatty acid synthesis by Bt2cAMP were reversed by lactate. Lactate also reversed the Bt2cAMP‐induced decrease in oxaloacetate and citrate concentration. The fact that lactate enters the pathway between glucose and oxaloacetate indicated that the site of inhibition by Bt, cAMP lies above pyruvate in the glycolytic pathway. Based on these and previous results it appears that, by inhibiting glycolysis, cAMP exerts long‐range effects on two major pathways in the chick hepatocyte, ketogenesis and fatty acid synthesis.

Gill citrate synthase from an abyssal fish

1.1. Citrate synthase, extracted from the gills of an abyssal fish, Antimora rostrata, and purified about 100-fold by ammonium sulfate fractionation and differential absorption to phosphocellulose, displays an unusual polymerization behaviour that encouraged an examination of its catalytic and regulatory properties. Like other vertebrate citrate synthases, the Antimora gill citrate synthase is under feedback inhibitory control by -ketoglutarate, ATP, and GTP.2.2. GTP and ATP are strictly competitive with respect to acetylCoA, but with respect to oxaloacetate, both the Km and the Vmax are reduced. Alpha-ketoglutarate greatly increases the Km for oxaloacetate, but does not affect the Vmax.3.3. AMP, in contrast, is an activator causing a small increase in the maximum velocity and a large increase in enzyme-oxaloacetate affinity.4.4. Although these regulatory properties are unaltered by pressure, the maximum velocity of the enzyme shows a pressure optimum at 200–250 atm, and this pressure activation is most pronounced when at least one of the substrates is at low (Km) levels.5.5. At high substrate levels or at high temperatures, the pressure sensitivity is reduced. In contrast, the Arrhenius activation energy is unaffected by pressure.6.6. These characteristics, coupled with unusually high specific activities, are all consistent with effective in vivo function despite the hostile physical environment of the abyss.

Some properties of the citrate synthase from the extreme halophile, Halobacterium cutirubrum.

1. Citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating), EC 4.1.3.7] was purified about 400-fold from the extreme halophile, Halobacterium cutirubrum, by a method involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and hydroxyapatite and gel filtration on Sephadex G-200. 2. The purified enzyme was best activated by high concentrations of KCl (3M); the chlorides of other cations and K+ salts of other anions (Br-, NO3-, SCN-) were less effective than KCl as activators. The enzyme was best stabilized by high concentrations of NaCl or KCl. Cold-lability was found in the presence of 3M-KCl, but not in the presence of NaCl at concentrations up to 5M. The results suggest that both the shielding of negative charges on the enzyme molecule and the stabilization of hydrophobic bonds by high KCl concentrations were required for maximum activity of the enzyme. 3. The double-reciprocal plots for acetyl-CoA or oxaloacetate at several concentrations of the co-substrate intersected at the abscissa in the presence of either KCl or NaCl, at either 1 or 3M. The Km for oxaloacetate increased about fivefold with the salt concentration, from 1 to 3M.

Effect of phenylpyruvate on enzymes involved in fatty acid synthesis in rat brain

1. The activities of, and the effects of phenylpyruvate on, citrate synthase (EC 4.1.3.7), acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase derived from the brains of 14-day-old and adult rats were investigated. 2. The brain citrate synthase from 14-day-old rats had a K(m) for oxaloacetate of 2.38mum and for acetyl-CoA of 16.9mum, and a V(max.) of 838nmol of acetyl-CoA incorporation/min per mg of mitochondrial protein. From adult rat brain this enzyme had a K(m) for oxaloacetate of 2.5mum and for acetyl-CoA of 16.6mum and a V(max.) of 1070nmol of acetyl-CoA incorporated/min per mg of mitochondrial protein. Phenylpyruvate inhibited the enzyme from adult and young rat brains in a competitive fashion with respect to acetyl-CoA, with a K(i) of 700mum. 3. The brain acetyl-CoA carboxylase from 14-day-old rats had a K(m) for acetyl-CoA of 21mum and a V(max.) of 0.248nmol/min per mg of protein, and from adult rats a K(m) for acetyl-CoA of 21mum and a V(max.) of 0.173nmol/min per mg of protein. The enzyme from young and adult rats required citrate (K(a)=3mm) for activation and were inhibited non-competitively by phenylpyruvate, with a K(i) of 10mm. 4. The brain fatty acid synthetase from 14-day-old rats had a K(m) for acetyl-CoA of 7.58mum and a V(max.) of 1.1 nmol of malonyl-CoA incorporated/min per mg of protein, and from adult rats a K(m) for acetyl-CoA of 4.9mum and a V(max.) of 0.48nmol of malonyl-CoA incorporated/min per mg of protein. Phenylpyruvate acted as a competitive inhibitor with respect to acetyl-CoA with a K(i) of 250mum for the enzyme from 14-day-old rats. 5. These results are discussed with respect to phenylketonuria, and it is suggested that the inhibition of the brain fatty acid synthetase and possibly the citrate synthetase by phenylpyruvate could explain the defective myelination characteristic of this condition.