Serine: glyoxylate, alanine:glyoxylate, and glutamate:glyoxylate aminotransferase reactions in peroxisomes from spinach leaves

Two different aminotransferases, that have glyoxylate as the amino acceptor, have specific activities of 1 to 2 mumol . min-1 . mg of protein-1 in the isolated peroxisomal fraction from spinach leaves. Their properties were evaluated after separation on a hydroxylapatite column. Both enzymes had a Km for glyoxylate of 0.15 mM and an amino acid Km of 2 to 3 mM. Reactions proceeded by a Ping Pong Bi Bi mechanism. Serine:glyoxylate aminotransferase was relatively specific for both substrates and could only be slightly reversed with 100 mM glycine, although the Ki of glycine was 33 mM. The glutamate:glyoxylate amino-transferase protein was equally active in catalyzing an alanine:glyoxylate aminotransferase reaction, but the reverse reactions with 100 mM glycine were hardly measureable, although the Ki (glycine) was 8.7 mM. Protection against hydroxylamine inhibition from reaction with pyridoxal phosphate was used to investigate the specificity of amino acid binding. Substrate amino acids protected at about the same concentration as their Km, while glycine protected at its Ki concentration. Thus, the nearly irreversible catalysis with glycine is not due to a failure to bind glycine. The significance of a peroxisomal alanine:glyoxylate aminotransferase activity has not been incorporated into schemes for the oxidative photosynthetic carbon cycle.     

Steady-state investigations of the mechanism of histidinol dehydrogenase.

Initial velocities of the histidinol dehydrogenase reaction (EC 1.1.1.23) were measured as a function of the concentrations of the substrates histidinol and NAD+ and in the presence and absence of the product NADH. The data are consistent with a Bi Uni Uni Bi Ping Pong mechanism. The kinetic constants of this mechanism were determined; Km for histidinol was found to be 14 microM and for NAD+ 0.7 mV; Ki for NAD+ was 0.4 mM.     

Studies of the specificity and kinetics of rat liver spermidine/spermine N1-acetyltransferase.

The substrate specificity and kinetic mechanism of spermidine N1-acetyltransferase from rat liver was investigated using a highly purified (18 000-fold) preparation from the livers of rats in which the enzyme was induced by treatment with carbon tetrachloride (1.5 ml/kg body wt. 6h before death). The enzyme catalysed the acetylation of spermidine, spermine, sym-norspermidine, sym-norspermine, N-(3-aminopropyl)-cadaverine, N1-acetylspermine, 3,3'-diamino-N-methyldipropylamine and 1,3-diaminopropane, but was inactive with putrescine, cadaverine, sym-homospermidine and N1-acetylspermidine. These results suggest that the enzyme is highly specific for the acetylation of a primary amino group that is separated by a three-carbon aliphatic chain from another nitrogen atom (i.e. the substrates are of the type H2N[CH2]3NHR). The maximal rates of acetylation of 1,3-diaminopropane and 3,3'-diamino-N-methyldipropylamine were much lower than the maximal rates with spermidine or sym-norspermidine as substrates, suggesting a preference for a secondary amino group bearing the aminopropyl group that is acetylated. The best substrates for acetylation were sym-norspermidine and sym-norspermine, which had Km values of about 10 micrograms and Vmax. values of about 2 mumol of product/min per mg of enzyme compared with Km of 130 microM and Vmax. of 1.3 mumol/min per mg for spermidine. N1-Acetylspermidine (the product of the reaction) and N8-acetylspermidine were weak inhibitors and were competitive with spermidine, having Ki values of about 6.6 mM and 0.4 mM respectively. N1-Acetylspermidine was a non-competitive inhibitor with respect to acetyl-CoA. CoA was also inhibitory to the reaction, showing non-competitive kinetics when either [acetyl-CoA] or [spermidine] was varied. These results suggest that the reaction occurs via an ordered Bi Bi mechanism in which spermidine binds first and N1-acetyl-spermidine is the final product to be released.     

The oxidation of methylglyoxal by mammalian pyruvate dehydrogenase

Mammalian pyruvate dehydrogenase actively catalyzed the oxidation of methylglyoxal to acetyl-CoA. The reaction was fully enzymatic with an estimated Km of 1.89 mM. On the other hand, methylglyoxal was a competitive inhibitor of the enzyme for pyruvate, the Ki being in the 1 mM range. The reaction was inhibited in the presence of HgCl2. The reaction products were quantitatively identified as acetyl-CoA and formic acid. A mechanism for the reaction is proposed.     

Regulation of ketogenesis. Mitochondrial acetyl-CoA acetyltransferase from rat liver: initial-rate kinetics in the presence of the product CoASH reveal intermediary plateau regions

The analysis of the initial-rate kinetics of the liver mitochondrial acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) in the direction of acetoacetyl-CoA synthesis under product inhibition was performed. 1. Acetyl-CoA acetyltransferase shows a hyperbolic response of reaction velocity to changes in acetyl-CoA concentrations with an apparent Km of 0.237 +/- 0.001 mM. 2. CoASH is a (non-competitive) product inhibitor with a Kis of 22.6 microM and shifts the apparent Km for acetyl-CoA to the physiological concentration of this substrate in mitochondria (S0.5 = 1.12 mM in the presence of 121 microM CoASH). 3. CoASH causes a transformation of the Michaelis-Menten kinetics into initial-rate kinetics with four intermediary plateau regions. 4. The product analogue desulpho-CoA triggers a negative cooperativity as to the dependence of the reaction velocity on the acetyl-CoA concentration. These product effects drastically desensitize the acetyl-CoA acetyltransferase in its reaction velocity response to the acetyl-CoA concentrations and simultaneously extend the substrate dependence range. Thus a control of acetoacetyl-CoA synthesis by the substrate is established over the physiological acetyl-CoA concentration range. We suggest that this control mechanism is the key in establishing the rates of ketogenesis.     

Purification and partial characterization of chicken liver acetyl coenzyme A: arylamine N-acetyltransferase

Arylamine acetyltransferase (EC 2.3.1.5) was purified 120-fold from chicken liver. The enzyme showed a rise in activity from pH 6.5 to 7.7 followed by a constant activity to about pH 8.6. The relative molecular weight of the enzyme was about 34,000. The apparent Km for acetyl-CoA was 13 microM with 4-nitroaniline as acetyl-acceptor. CoA was a noncompetitive inhibitor relative to acetyl-CoA with apparent Ki value of 110 microM. With 4-methylaniline as substrate, arylamine acetyltransferase activity in pigeon liver was about 8 times greater than in chicken liver, and about 40 times greater than in rabbit.     

Effect of environmental temperature on the kinetic properties of goldfish brain choline acetyltransferase

1. Michaelis constants of goldfish brain choline acetyltransferase were found to depend on the concentration of the second substrate present and on the temperature to which the fish had been adapted. 2. Primary plots constructed from results obtained with enzyme prepared from cold-adapted or warm-adapted fish indicated that synthesis of acetylcholine took place by a sequential mechanism. 3. The affinity of choline acetyltransferase for acetyl-CoA was about 100 times that for choline irrespective of whether the enzyme had been prepared from warm-adapted or cold-adapted fish. 4. The maximum rate at which choline acetyltransferase synthesized acetylcholine and the energy of activation for this synthesis remained independent of the previous environmental temperature of the fish. 5. The affinity of choline acetyltransferase for choline and acetyl-CoA showed a complex dependence on temperature. The affinity of the enzyme from cold-adapted fish for substrates increased as the incubation temperature was lowered, whereas that of the enzyme from warm-adapted fish first increased and then decreased. 6. The maximum affinity of choline acetyltransferase for both substrates, from both cold-adapted and warm-adapted fish, occurred at temperatures that corresponded approximately to the respective environmental temperatures of the fish. 7. These changes in enzyme affinity for substrates are not thought to be due to the presence of isoenzymes. Their adaptive significance is unknown, but it could be connected with the maintenance of the enzyme in a stable form.     

Purification and some properties of phosphoenolpyruvate carboxylase from Brevibacterium flavum and its aspartate-overproducing mutant

Phosphoenolpyruvate (PEP) carboxylases (PC) were purified from a wild strain and an aspartate-producing mutant of Brevibacterium flavum to electrophoretic homogeneity. The molecular weights of the enzymes were determined to be 4.1 X 10(5) by the gel-filtration technique. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme gave only one protein band with a molecular weight of 1.07 X 10(5). The enzyme was labile and stabilized by substrate PEP, activators, metallic cofactors, an allosteric inhibitor and ammonium sulfate. The mechanism for the PC reaction was rapid equilibrium random Bi Bi with a dead end complex, enzyme-bicarbonate-Pi. The KmS for PEP and bicarbonate were 2.5 and 0.63 mM, respectively, and the apparent KmS were not affected by the secondary substrate concentrations. Dissociation constants for Pi of enzyme-Pi and the dead end complex were 5.0 and 16 mM, respectively. Aspartate inhibition was completely competitive with both the substrates, PEP and bicarbonate, with an inhibitor constant of 0.044 mM. An activator, acetyl-CoA, did not alter the apparent Km for bicarbonate but decreased that for PEP. The activator constants for the enzyme-PEP complex and free enzyme were 6.3 and 40 microM, respectively. Double reciprocal plots of reaction rate against PEP concentration were not linear at lower PEP concentrations. Hill coefficients for PEP were 1.6 in the absence of any effectors, 1.0 in the presence of acetyl-CoA, and 2.3 in the presence of aspartate. As to the mutant enzyme, only the inhibitor constant for aspartate was increased, being 0.18 mM, but other constants, coefficients, as described above, and specific activity were almost the same as those of the wild-type enzyme.     

Kinetics for formate dehydrogenase of Escherichia coli formate-hydrogenlyase

Kinetic parameters of the selenium-containing, formate dehydrogenase component of the Escherichia coli formate-hydrogenlyase complex have been determined with purified enzyme. A ping-pong Bi Bi kinetic mechanism was observed. The Km for formate is 26 mM, and the Km for the electron-accepting dye, benzyl viologen, is in the range 1-5 mM. The maximal turnover rate for the formate-dependent catalysis of benzyl viologen reduction was calculated to be 1.7 x 10(5) min-1. Isotope exchange analysis showed that the enzyme catalyzes carbon exchange between carbon dioxide and formate in the absence of other electron acceptors, confirming the ping-pong reaction mechanism. Dissociation constants for formate (12.2 mM) and CO2 (8.3 mM) were derived from analysis of the isotope exchange data. The enzyme catalyzes oxidation of the alternative substrate deuterioformate with little change in the Vmax, but the Km for deuterioformate is approximately three times that of protioformate. This implies formate oxidation is not rate-limiting in the overall coupled reaction of formate oxidation and benzyl viologen reduction. The deuterium isotope effect on Vmax/Km was observed to be approximately 4.2-4.5. Sodium nitrate was found to inhibit enzyme activity in a competitive manner with respect to formate, with a Ki of 7.1 mM. Sodium azide is a noncompetitive inhibitor with a Ki of about 80 microM.     

Acetylation of 13-sophorosyloxydocosanoic acid by an acetyltransferase purified from Candida bogoriensis.

Acetyl-coenzyme A: 13-sophorosyloxydocosanoic acid (Glc2HDA) acetyltransferase was purified 14-fold in low yield from Candida bogoriensis cells. The enzyme catalyzes acetylation of the 6' and 6" positions of the sophorosyl group, producing the 13-[2'-O-beta-D-glucopyranosyl-beta-D-glucopyranosyloxy]-docosanoic acid 6',6"-diacetate (Ac2Glc2HDA) and monoacetate (AcGlc2HDA) in a product ratio of 5:1. Neither the purification steps nor heat denaturation studies indicated separation of the first and second acetylation steps. The acetyltransferase has a molecular weight of about 500,000 as determined by gel filtration on a Sepharose 4-B column. It shows a pH optimum range from 7 to 9, is strongly inhibited by 1 mM concentrations of the sulfhydryl reagents N-ethylmaleimide, p-hydroxymercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoic acid), but only partly inhibited by 10 mM iodoacetamide. It has an apparent Km of 30 muM for acetyl-CoA, utilizes propionyl-CoA at 45% the rate of acetyl-CoA, and utilizes longer chain acyl-CoA derivatives much less efficiently. The critical micelle concentrations of the C. bogoriensis glycolipids in pH 7.7 phosphate buffer were estimated by pinacyanol chloride binding as follows: Glc2HDA, 50 mum; AcGlc2HDA, 30 muM; Ac2Glc2HDA, 12 muM. The Stokes radius of Ac2Glc2HDA micelles was 22 A as estimated by gel filtration on Bio-Gel P-150. Glc2HDA was a much better acceptor than its methyl ester in the acetyltransferase assay. A plateau in the Glc2HDA saturation curve at 50 muM and a corresponding break in the reciprocal plot at this concentration indicate the enzyme utilizes the monomeric form of this lipid as substrate.     

Phenylalanine Transport Across the Blood-Brain Barrier as Studied with the In Situ Brain Perfusion Technique

Unidirectional L-phenylalanine transport into six brain regions of pentobarbital-anesthetized rats was studied using the in situ brain perfusion technique. This technique allows both accurate measurements of cerebrovascular amino acid transport and complete control of perfusate amino acid composition. L-Phenylalanine influx into the brain was sodium independent and could be described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants in the parietal cortex equaled 6.9 X 10(-4) mumol/s/g for Vmax, 0.011 mumol/ml for Km, and 1.8 X 10(-4) ml/s/g for KD during perfusion with fluid that did not contain competing amino acids. D-Phenylalanine competitively inhibited L-phenylalanine transport with a Ki approximately 10-fold greater than the Km for L-phenylalanine. There were no significant regional differences in Km, KD, or Ki, whereas Vmax was significantly greater in the cortical lobes than in the other brain regions. L-Phenylalanine influx during plasma perfusion was only 30% of that predicted in the absence of competing amino acids. Competitive inhibition increased the apparent Km during plasma perfusion by approximately 20-fold, to 0.21 mumol/ml. These data provide accurate new estimates of the kinetic constants that describe L-phenylalanine transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer site affinity (1/Km) for L-phenylalanine is three- to 12-fold greater than previously estimated in either awake or anesthetized animals.     

Effects of dietary nutrients on substrate and effector levels of lipogenic enzymes, and lipogenesis from tritiated water in rat liver

When fasted rats were refed for 4 days with a carbohydrate and protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate), the effects of dietary nutrients on the fatty acid synthesis from injected tritiated water, the substrate and effector levels of lipogenic enzymes and the enzyme activities were compared in the livers. In the carbohydrate diet group, although acetyl-CoA carboxylase was much induced and citrate was much increased, the activity of acetyl-CoA carboxylase extracted with phosphatase inhibitor and activated with 0.5 mM citrate was low in comparison to the carbohydrate and protein diet group. The physiological activity of acetyl-CoA carboxylase seems to be low. In the protein diet group, the concentrations of glucose 6-phosphate, acetyl-CoA and malonyl-CoA were markedly higher than in the carbohydrate and protein group, whereas the concentrations of oxaloacetate and citrate were lower. The levels of hepatic cAMP and plasma glucagon were high. The activities of acetyl-CoA carboxylase and also fatty acid synthetase were low in the protein group. By feeding fat, the citrate level was not decreased as much as the lipogenic enzyme inductions. Comparing the substrate and effector levels with the Km and Ka values, the activities of acetyl-CoA carboxylase and fatty acid synthetase could be limited by the levels. The fatty acid synthesis from tritiated water corresponded more closely to the acetyl-CoA carboxylase activity (activated 0.5 mM citrate) than to other lipogenic enzyme activities. On the other hand, neither the activities of glucose-6-phosphate dehydrogenase and malic enzyme (even though markedly lowered by diet) nor the levels of their substrates appeared to limit fatty acid synthesis of any of the dietary groups. Thus, it is suggested that under the dietary nutrient manipulation, acetyl-CoA carboxylase activity would be the first candidate of the rate-limiting factor for fatty acid synthesis with the regulations of the enzyme quantity, the substrate and effector levels and the enzyme modification.     

Substrate binding to chloramphenicol acetyltransferase: evidence for negative cooperativity from equilibrium and kinetic constants for binary and ternary complexes

Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol by a ternary complex mechanism with a rapid equilibrium and essentially random order of addition of substrates. Such a kinetic mechanism for a two-substrate reaction provides an opportunity to compare the affinity of enzyme for each substrate in the binary complexes (1/Kd) with corresponding values (1/Km) for affinities in the ternary complex where any effect of the other substrate should be manifest. The pursuit of such information for CAT involved the use of four independent methods to determine the dissociation constant (Kd) for chloramphenicol in the binary complex, techniques which included stopped-flow measurements of on and off rates, and a novel fluorometric titration method. The binary complex dissociation constant (Kd) for acetyl-CoA was measured by fluorescence enhancement and steady-state kinetic analysis. The ternary complex dissociation constant (Km) for each substrate (in the presence of the other) was determined by kinetic and fluorometric methods, using CoA or ethyl-CoA to form nonproductive ternary complexes. The results demonstrate an unequivocal decrease in affinity of CAT for each of its substrates on progression from the binary to the ternary complex, a phenomenon most economically described as negative cooperativity. The binary complex dissociation constants (Kd) for chloramphenicol and acetyl-CoA are 4 microM and 30 microM whereas the corresponding dissociation constants in the ternary complex (Km) are 12 microM and 90 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyruvate dehydrogenase complex of ascites tumour. Activation by AMP and other properties of potential significance in metabolic regulation.

1. AMP is an activator of the pyruvate dehydrogenase complex of the Ehrlich--Lettré ascites tumour, increasing its V up to 2-fold, with Ka of 40 microM at pH 7.4. This activation appears to be an allosteric effect on the decarboxylase subunit of the complex. 2. The pyruvate dehydrogenase complex has a Km for pyruvate within the range 17--36 microM depending on the pH, the optimum pH being approx. 7.4, with a V of approx. 0.1 unit/g of cells. The rate-limiting step is dependent on the transformation of the enzyme--substrate complex. The Km for CoA is 15 microM. The Km for NAD+ is 0.7 mM for both the complex and the lipoamide dehydrogenase. The complex is inhibited by acetyl-CoA competitively with CoA; the Ki is 60 microM. The lipoamide dehydrogenase is inhibited by NADH and NADPH competitively with NAD+, with Ki values of 80 and 90 microM respectively. In the reverse reaction the Km values for NADH and NADPH are essentially equal to their Ki values for the forward reaction, the V for the latter being 0.09 of that of the former. Hence the reaction rate of the complex in vivo is likely to be markedly affected by feedback isosteric inhibition by reduced nicotinamide nucleotides and possibly acetyl-CoA.     

Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase

The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.     

4-Aminobutyrate:2-oxoglutarate aminotransferase from Candida. Purification and properties

An enzyme which catalyzes the transamination of 4-aminobutyrate with 2-oxoglutarate was purified 588-fold to homogeneity from Candida guilliermondii var. membranaefaciens, grown with 4-aminobutyrate as sole source of nitrogen. An apparent relative molecular mass of 107,000 was estimated by gel filtration. The enzyme was found to be a dimer made up of two subunits identical in molecular mass (Mr 55,000). The enzyme has a maximum activity in the pH range 7.8-8.0 and a temperature optimum of 45 degrees C. 2-Oxoglutarate protects the enzyme from heat inactivation better than pyridoxal 5'-phosphate. The absorption spectrum of the enzyme exhibits two maxima at 412 nm and 330 nm. The purified enzyme catalyzes the transamination of omega-amino acids; 4-aminobutyrate is the best amino donor and low activity is observed with beta-alanine. The Michaelis constants are 1.5 mM for 2-oxoglutarate and 2.3 mM for 4-aminobutyrate. Several amino acids, such as alpha,beta-alanine and 2-aminobutyrate, are inhibitors (Ki = 38.7 mM, Ki = 35.5 mM and Ki = 33.2 mM respectively). Propionic and butyric acids are also inhibitors (Ki = 3 mM and Ki = 2 mM).     

Trichomonas vaginalis: characterization of its glutamate dehydrogenase

An NADP-linked glutamate dehydrogenase (EC 1.4.1.4) was found in the soluble fraction of Trichomonas vaginalis. Its molecular weight was about 230,000 (gel filtration). The enzyme, partially purified by diafiltration and hydroxyapatite column chromatography, was heat stable (1 hr at 57 C). It catalyzed both the amination of alpha-ketoglutarate (mean Km 0.6 mM) and the deamination of glutamate (mean Km 1.2 mM) The optimum pH of the amination reaction was 6.7, and that of the deamination reaction was 8. Glutamate was a competitive inhibitor of the amination reaction (mean Ki 5.6 mM) and alpha-ketoglutarate a partially competitive inhibitor of the deamination reaction (mean Ki 0.45 mM). Both guanosine and inosine diphosphates (1 mM) increased the Km alpha-ketoglutarate fivefold (mean Ki's 0.3 and 0.4 mM, respectively). Guanosine diphosphate reduced the Km glutamate 40%. Adenosine di- and triphosphate (1 mM) were ineffective. Because the amination reaction displayed substrate inhibition, guanosine and inosine diphosphates were potent natural inhibitors, and ammonia released by deamination reactions would tend to raise pH (amination operative at acid pH), we hypothesize that the deamination reaction may predominate in the living organism.     

Substrate inhibition of pigeon liver fatty acid synthetase and optimum assay conditions for over-all synthetase activity

The effects of the substrates acetyl-CoA, malonyl-CoA, and NADPH on the activity of pigeon liver fatty acid synthetase have been studied over a wide range of concentrations. Double-reciprocal coordinate plots for each of the substrates have been found to be linear at low concentrations. At higher concentrations two of the substrates, acetyl-CoA and malonyl-CoA, inhibit the rate of fatty acid synthesis. This double substrate inhibition is apparently of a competitive type. Inhibition by acetyl-CoA is very strong as compared to that by malonyl-CoA. At a 4:1 ratio of acetyl- to malonyl-CoA, inhibition is about 75%, whereas at a 4:1 ratio of malonyl- to acetyl-CoA fatty acid synthesis proceeds at the maximum rate.These results are consistent with the hypothesis that a competition between acetyl-CoA and malonyl-CoA occurs for the occupany of the 4′- phosphopantetheine site, a prosthetic group of the synthetase complex, and possibly also for the hydroxyl binding site (or sites). The relative concentrations of these substrates and the binding constants for each then determine whether these sites are occupied by acetyl or malonyl groups, and whether inhibition of fatty acid synthesis occurs. Based on our results, assays for pigeon liver fatty acid synthetase activity should be conducted at substrate concentrations of 15 μm, 60 μm, and 100 μm for acetyl-CoA, malonyl-CoA, and NADPH, respectively.