Sarvagya S. Katiyar

Differential interaction of fatty acids and fatty acyl CoA esters with the purified/reconstituted brown adipose tissue mitochondrial uncoupling protein

Proteoliposomes containing highly purified uncoupling protein generated by a modified purification/reconstitution procedure carried out active GDP dependent proton conductance. It was further established that long chain acyl CoA esters as well as fatty acids stimulated proton influx by the uncoupling protein, and, moreover, that the acyl CoA esters were partially effective in overcoming the inhibition by GDP. GDP binding to the purified uncoupling protein was inhibited by acyl CoA esters but not fatty acids. Phenylglyoxal which prevents GDP binding to the uncoupling protein eliminated the acyl CoA but not the fatty acid effect on proton conductance. These results substantiate the fact that nucleotides and acyl CoA esters act at the same regulatory site on the uncoupling protein, whereas, fatty acids act at a separate site. The properties of the purified/reconstituted uncoupling protein confirm they are identical to those inherent in brown adipose tissue mitochondria.

Synthesis of fatty acids from malonyl-CoA and NADPH by pigeon liver fatty acid synthetase

Abstract Pigeon liver fatty acid synthetase has been found to catalyze the formation of palmitic acid from malonyl-CoA and NADPH in the absence of acetyl-CoA. Radio-chemical and spectral assays show that the activity of the complex in the absence of acetyl-CoA is about 25–30% of the activity in the presence of this compound. Initial velocities were determined for a series of reactions in which the malonyl-CoA concentration was varied over a range of 5–200 μ m at a fixed NADPH concentration of 100μ m and vice versa. No inhibitory effects of one substrate over the other were found. However, when the synthesis of fatty acids was studied in the presence of acetyl-CoA, a significant inhibitory effect of malonyl-CoA was observed. It has also been shown that the fatty acid synthetase synthesizes triacetic lactone from malonyl-CoA in the absence of NADPH and acetyl-CoA. No evidence was obtained for the direct decarboxylation of malonyl-CoA to acetyl-CoA in this reaction. Hence it is proposed that decarboxylation of the malonyl moiety bound covalently to 4′-phosphopantetheine occurs to yield acetyl-4′-phosphopantetheine. Further, it is proposed that the acetyl moiety of the latter compound is transferred to the cysteine site of the enzyme complex and that fatty acid synthesis proceeds in the presence of NADPH as proposed by Phillips et al. [ Arch. Biochem. Biophys. 138 , 380 (1970) ]. In the absence of NADPH triacetic lactone is formed.

Adenosine 5'-triphosphate stimulation of the activity of a partially purified phytoene synthetase complex.

Summary An enzyme complex capable of converting isopentenyl pyrophosphate to phytoene has been isolated from an acetone powder of tomato fruit plastids and partially purified. This complex, approximately 200,000 Daltons in molecular weight, is devoid of squalene synthetase activity. Mn++ is required for enzyme activity, but no other cofactors have been identified as being essential. However, a 6 to 7-fold stimulation in activity is effected by ATP (1.3 mM). Since there is no evidence that ATP participates directly in the synthesis of phytoene from isopentenyl pyrophosphate, it is suggested that this nucleotide may be an allosteric effector of the reaction.

Role of cysteine and 4'-phosphopantetheine in the inactivation of pigeon liver fatty acid synthetase by S-(4-bromo-2,3-dioxobutyl)-coenzyme A.

Abstract S-(4-bromo-2,3-dioxobutyl)-CoA has been used as an inhibitor of fatty acid synthetase from pigeon liver. This affinity label selectively and irreversibly inhibits the acetyl transacylase and β-ketoacyl synthetase reactions of this multienzyme complex. Binding studies with [3H]-labeled bromodioxobutyl-CoA have established that four mol of the inhibitor are bound per mol of the enzyme complex, and that the radioactivity of this compound is covalently bound to cysteine and 4′-phosphopantetheine moieties. Other partial reactions of fatty acid synthesis are unaffected by bromodioxobutyl-CoA.

Evidence for catalytic site cysteine and histidine by chemical modification of β-hydroxy-β-methylglutaryl-coenzyme A reductase

Summary S-(4-Bromo-2,3-dioxobutyl)-coenzyme A inactivates both yeast and rat liver β-hydroxy-β-methylglutaryl-coenzyme A reductase. The inactivation is irreversible, complete in 15 s, and proportional to the concentration of the reagent. β-Hydroxy-β-methylglutaryl-CoA provides protection against inactivation, whereas NADPH does not. Inactivation is attributed to reaction with an essential cysteine at the β-hydroxy-β-methylglutaryl-CoA binding site. Experiments with other active site-directed reagents confirm the involvement of a cysteine and support the presence of an active-site histidine, but rule out the participation of arginine or serine.

Affinity purification of anti-pigeon liver fatty acid synthetase immunoglobulin and comparative immunoreactivity of the catalytic reactions☆

Abstract Rabbit anti-pigeon liver fatty acid synthetase antibody was prepared by affinity chromatography on Sepharose-fatty acid synthetase to near monospecificity (98% or more) as shown by immunodiffusion plates and rocket immunoelectrophoresis. Immunotitrations of the highly purified monospecific antibody against the overall activity and partial activities of fatty acid synthetase were then carried out. Only 6 mol of antibody/mol of enzyme was required to inactivate overall fatty acid synthetase activity and the condensation reaction, while 12 to 18 mol were required to partially inactivate the β-ketoacyl reductase and the malonyl- and acetyl-CoA transferases. Palmitoyl-CoA thioesterase (deacylase) activity was not inhibited by the antibody. The degree of inactivation of the partial reactions by antibody was not affected by dissociation of the fatty acid synthetase. Immunoprecipitation of the enzyme indicated that there are approximately 35 immunoreactive sites on the fatty acid synthetase molecule. The possible implications of these results to an understanding of the structural organization of pigeon liver fatty acid synthetase and its antigenic determinants are discussed.

The involvement of a lysine residue at the active site of the enoyl reductase of pigeon liver fatty acid synthetase.

The reaction of pyridoxal-5′-phosphate with lysine residues of pigeon and rat liver fatty acid synthetases has been investigated. The enzyme is reversibly inactivated by this compound inasmuch as overall and enoyl reductase activities are lost as a function of time. β-Ketoacyl reductase activity and other component activities of the enzyme are not affected. Spectrophotometric assays, as well as amino acid analysis of the acid hydrolysate of NaB 3 H 4 -reduced enzyme-pyridoxal phosphate complex, have established that the inactivation of the enzyme is due to Schiff base formation with e-amino groups of lysine residues that are essential for the enoyl reductase component activity of fatty acid synthetases.

Kinetic study of the synthesis of fatty acids from malonyl-CoA and NADPH by pigeon liver fatty acid synthetase.

Abstract In the absence of acetyl-CoA pigeon liver fatty acid synthetase catalyzes the formation of palmitic acid from malonyl-CoA and NADPH [ Katiyar, Briedis, and Porter (1974) Arch. Biochem. Biophys. 162 , 412–420 ]. A kinetic analysis of this reaction is reported in the present paper. Initial velocity double reciprocal plots, in the absence of products, give a parallel pattern irrespective of which substrate is varied. No substrate inhibition by either substrate, malonyl-CoA or NADPH, is observed. The product inhibition pattern of NADP + is competitive with respect to NADPH and uncompetitive with respect to malonyl-CoA; whereas coenzyme A gives a competitive pattern vs malonyl-CoA and an uncompetitive pattern vs NADPH. These kinetic patterns are compatible with the 7-site ping-pong mechanism established earlier by us for the overall fatty acid synthetase reaction in the presence of all three substrates [ Katiyar, Cleland, and Porter (1975) J. Biolo. Chem. , 250 , 2709–2717 ].

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

Abstract 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 μ m , 60 μ m , and 100 μ m for acetyl-CoA, malonyl-CoA, and NADPH, respectively.

Liberation, purification, and properties of thioesterase component of pigeon liver fatty acid synthetase complex

Proteolysis of pigeon liver fatty acid synthetase with elastase results in the quantitative cleavage of the thioesterase component from the enzyme complex. This thioesterase component is two or three times more active catalytically in the isolated state than in the native fatty acid synthetase, and its activity is not affected by the presence or absence of reducing thiols. The proteolytically cleaved thioesterase is separated from the core enzyme in one step by size-exclusion chromatography on a Sephadex G-75 column. The peptide obtained by gel permeation is homogeneous with respect to size and charge, as shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. Size-exclusion chromatography on Bio-Gel A 0.5 m and Sephadex G-75 columns, sucrose density gradient ultracentrifugation, and N-terminal amino acid analysis also indicate that the proteolytically cleaved thioesterase is homogeneous. The sedimentation coefficient of the thioesterase is approximately 2.9 S. Proteolytic cleavage with elastase also quantitatively releases the [1,3-14C]- or [1,3-3H]diisopropylphosphofluoridate-labeled thioesterase component from the correspondingly labeled fatty acid synthetase. Binding studies with 14C- or 3H-labelled diisopropylphosphofluoridate and fatty acid synthetase show that 2 mol of the label are bound per mol of the enzyme when complete loss of fatty acid-synthesizing activity occurs. The molecular weight of the thioesterase component is estimated to be 36000 by size-exclusion chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis.