Separation of pigeon liver apo- and holo- fatty acid synthetases by affinity chromatography.

Apo- and holo-fatty acid synthetases of pigeon liver were separated by affinity gel chromatography under conditions similar to, but not identical to, those used in separating subunits I and II of [¹⁴C]pantetheine-labeled fatty acid synthetase complex [Lornitzo $\text{et al.}$, J. Biol. Chem. $\text{249}$, 1654 (1974)]. When [¹⁴C]pantetheine-labeled fatty acid synthetases were separated, the enzymatically active holo form contained all of the [¹⁴C] label. Incubation of the apo-pigeon liver fatty acid synthetase complex with CoA, ATP and a partially purified pigeon liver soluble enzyme system, from which fatty acid synthetase had been removed, resulted in the formation of holo-enzyme. Activation of apo-fatty acid synthetase could also be achieved by replacing the apo-(4′-phosphopantetheine-less) acyl carrier protein with holo-acyl carrier protein. It is evident, therefore, that the inactive apo-fatty acid synthetase lacks a 4′-phosphopantetheine group.     

Differential effects of Mg2+ on various hydrolytic and synthetic activities of multifunctional glucose-6-phosphatase

The adaptive synthesis of fatty acid synthetase in the livers of rats fed a fat-free diet following 48 hr of fasting has been studied using immunochemical methods. The development of fatty acid synthetase activity during adaptive synthesis occurs about 3 hr following feeding, whereas the synthesis of material precipitable by anti-fatty acid synthetase serum, as judged by the incorporation of ³H-labeled amino acids into the immunoprecipitate, commenced within 1 hr. Extracts of liver of rats fed a fat-free diet for 1–3 hr following fasting contain increasing amounts of material which competes with purified fatty acid synthetase for antibody binding sites, even though they have no fatty acid synthetase activity. This suggests the presence of enzymatically inactive precursors of fatty acid synthetase in the liver extracts. The incorporation of [¹⁴C]pantothenate into fatty acid synthetase during adaptive synthesis follows the same pattern as the development of enzyme activity, indicating that these enzymatically inactive precursors of fatty acid synthetase may represent an apoenzyme which is converted to the enzymatically active holoenzyme by the incorporation of the 4′-phosphopantetheine prosthetic group. The subcellular site of synthesis of fatty acid synthetase was shown to be in the pool of polysomes that are not membrane bound, rather than in the rough endoplasmic reticulum.     

Coenzyme A: fatty acid synthetase apoenzyme 4'-phosphopantetheine transferase in yeast

A mixture of two pantetheine-free mutant fatty acid synthetases was dissociated and recombined $\text{in}$$\text{vitro}$ to form a hybrid apoenzyme complex. $\text{In}$$\text{vivo}$ the corresponding $\text{Saccharomyces}$$\text{cerevisiae}$$\text{fas}$-mutants exhibit interallelic complementation when crossed with each other and the enzyme synthesized in the resulting diploid contains pantetheine and exhibits overall fatty acid synthetase activity. Accordingly, the hybrid apoenzyme formed $\text{in}$$\text{vitro}$ could be activated to holo-fatty acid synthetase when incubated with coenzyme A and a partially purified yeast cell extract. The enzyme coenzyme A: fatty acid synthetase apoenzyme 4′-phosphopantetheine transferase has thus been identified in yeast. Further studies on the mechanism of fatty acid synthetase holoenzyme formation will now be possible.     

Isolation,purification, and properties of mammalian and avian liver and yeast fatty acid synthetase acyl carrier proteins

Acyl carrier proteins were isolated from rat, human, pigeon, and chicken liver and yeast fatty acid synthetase complexes. These proteins were separated from the other proteins of subunit I of each complex by ultrafiltration after dialysis of subunit I for 3 h against low ionic strength buffer [Qureshi et al. (1974) Biochem. Biophys. Res. Commun.60, 158–165]. Subunit I of each fatty acid synthetase was previously separated from subunit II by affinity chromatography on Sepharose ?-aminocaproyl pantetheine and subsequent sucrose density gradient centrifugation. The separated acyl carrier proteins were then subjected to gel filtration on a Sephadex G-50 column. The proteins obtained from each fatty acid synthetase were homogeneous with respect to size and charge on gel filtration, paper and disc gel electrophoresis, and chromatography on diethylaminoethyl-cellulose. The physical properties and the ability to accept acetyl and malonyl groups from acetyl- and malonyl-CoA in the presence of transacylase were similar to those of Escherichia coli acyl carrier protein. These proteins ranged in molecular weight from 7500 to 10,000. Each of the acyl carrier proteins showed the presence of β-alanine and each yielded acetyl- and malonyl-A₁ and A₂ peptic peptides, thus indicating the presence of a 4′-phosphopantetheine prosthetic group in each. They differed somewhat from each other in amino acid composition, but each had a high number of negatively charged (aspartate and glutamate) amino acid residues.     

Adaptive synthesis of rat liver fatty acid synthetase: evidence for in vitro formation of active enzyme from inactive protein precursors and 4'-phosphopantetheine

Evidence is presented in support of the hypothesis that an important step in the adaptive synthesis of fatty acid synthetase is the conversion of inactive enzyme precursors to active enzyme via the incorporation of the 4′-phosphopantetheine prosthetic group. Fatty acid synthetase activity was generated $\text{in vitro}$ when CoA or $\text{E. coli}$ acyl carrier protein was incubated with enzymatically inactive extracts from livers of rats fed a fat-free diet for 0–5 hr following starvation, and a factor present in liver extracts from rats refed for more than 6 hr. When (¹⁴C)-CoA, labelled in the pantetheine moiety, was used in the above system, radioactivity was incorporated into a protein bound form, from which it could be released by mild alkaline hydrolysis.     

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

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.     

Occurrence of apoAcyl carrier protein and an S-alkylation-resistant form of holoAcyl carrier protein in Escherichia coli.

The 4′-phosphopantetheine prosthetic group of holoacyl carrier protein (holoACP) in Escherichia coli turns over independently of the apoprotein, due to the activities of holoACP hydrolase and holoACP synthetase. There is no measurable pool of apoACP in pantothenate-supplemented cells of a pantothenate-requiring mutant, but extended incubation on deficient medium, with exhaustion of cellular coenzyme A (CoA), leads to slow accumulation of the apoprotein. It is concluded that, although the activities of the synthetase and hydrolase are about equal in crude extracts, in the cells an excess synthetase activity maintains ACP completely as holoACP unless cells are artifically depleted of CoA, the donor of the 4′-phosphopantetheine group. About 20% of the holoACP in normal cells was designated as “holoACP esters,” being resistant to S-alkylation unless first treated with neutral hydroxylamine; this proportion increased to about 80% in pantothenate starvation. A preliminary attempt to identify acyl portions from this material was unsuccessful. The proportion of this material was not elevated in other strains under conditions which show feedback inhibition of fatty acid biosynthesis in vivo.     

Acetyl-Coenzyme A carboxylase. Role of the prosthetic group in enzyme polymerization.

Apo-(acetyl-CoA carboxylase) completely free from the holoenzyme was prepared from biotin-deficient rat adipose tissue by using affinity chromatography. The apoenzyme does not aggregate under conditions favouring the transition of the holoenzyme to the polymeric form. Such transition is possible after the conversion of the apoenzyme into the holoenzyme in vitro, thus demonstrating the requirement of the prosthetic biotinyl group for enzyme activation.     

Circular dichroism of holo- and apoprotocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

Circular dichroism studies have been carried out on both apo- and holoprotocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa, in the absence and presence of competitive inhibitors, protocatechualdehyde and 4-nitrocatechol. The apo- and holoenzyme showed identical spectra in the ultraviolet region between 200 and 250 nm (peptide back bone region), but the low intensity negative bands at 330 and 480 nm of the holoenzyme were completely absent in the apoenzyme. On the side chain region, the positive ellipticity peaks of the holoenzyme change into a lower intensity and broader band indicating the participation of aromatic amino acid residues in the primary binding of iron ion. Under anaerobic conditions, spectral changes were evident in the side chain region for the binary complexes of both the holo- and the apoenzyme with protocatechuate. The presence of iron in the holoenzyme results in an increase in positive ellipticity between 290 and 320 nm. Either with or without the iron, the enzyme protein binds protocatechuate and has a greater positive circular dichroism increase at 240-260 nm. CD difference spectra indicate that the modes of binding to form the binary complexes of holo- or apoenzyme with either substrates or competitive inhibitors are different. The bound iron ion stimulates binding. Spectral changes of the holoenzyme in the aromatic region were also observed in different pH environments of lower enzymatic activity. It is still not established whether these aromatic residues play an active or passive role in the binding of iron and/or substrates and inhibitors.     

The isolation of acyl carrier protein from the pigeon liver fatty acid synthetase complex1 II

A low molecular weight protein of less than 10, 000 Daltons has been isolated from Subunit I (β-ketoacyl thioester reductase) of the pigeon liver fatty acid synthetase complex and purified to homogeneity. This protein contains all of the [¹⁴C]-labeled pantetheine incorporated into the fatty acid synthetase on injection of [¹⁴C]-labeled pantetheine into pigeons. It also has one β-alanine and one sulfhydryl group. This protein is an acceptor of an acetyl group from acetyl-CoA and a malonyl group from malonyl-CoA in the presence of Subunit II (transacylase). In these respects it is very similar to $\text{E. coli}$ acyl carrier protein.     

Regulatory properties of brain glutamate decarboxylase

1. Glutamate decarboxylase is a focal point for controlling gamma-aminobutyric acid (GABA) synthesis in brain. Several factors that appear to be important in the regulation of GABA synthesis have been identified by relating studies of purified glutamate decarboxylase to conditions in vivo. 2. The interaction of glutamate decarboxylase with its cofactor, pyridoxal 5'-phosphate, is a regulated process and appears to be one of the major means of controlling enzyme activity. The enzyme is present in brain predominantly as apoenzyme (inactive enzyme without bound cofactor). Studies with purified enzyme indicate that the relative amounts of apo- and holoenzyme are determined by the balance in a cycle that continuously interconverts the two. 3. The cycle that interconverts apo- and holoenzyme is part of the normal catalytic mechanism of the enzyme and is strongly affected by several probable regulatory compounds including pyridoxal 5'-phosphate, ATP, inorganic phosphate, and the amino acids glutamate, GABA, and aspartate. ATP and the amino acids promote apoenzyme formation and pyridoxal 5'-phosphate and inorganic phosphate promote holoenzyme formation. 4. Numerous studies indicate that brain contains multiple molecular forms of glutamate decarboxylase. Multiple forms that differ markedly in kinetic properties including their interactions with the cofactor have been isolated and characterized. The kinetic differences among the forms suggest that they play a significant role in the regulation of GABA synthesis.     

On the significance of the prosthetic group composition of citrate lyase.

1. Klebsiella aerogenes contains two different acyl carrier proteins, one specific for citrate lyase, the other for fatty acid synthetase. 2. The acyl carrier protein of fatty acid synthetase from K. aerogenes was isolated and compared with the corresponding protein from Escherichia coli and with the acyl carrier protein of citrate lyase from K. aerogenes. 3. As judged from prosthetic group compositions as well as amino acid and fingerprint analyses, the acyl carrier proteins of the two fatty acid synthetases are nearly identical but different from that of citrate lyase from K. aerogenes. 4. Therefore, the different prosthetic groups alone cannot be responsible for the different specificities of the acyl carrier proteins of fatty acid synthetase and citrate lyase in K. aerogenes. 5. The prosthetic group of citrate lyase, phosphoribosyl dephospho-CoA, apparently represents no incidental, phosphopantetheine-replacing aberration. The requirement of citrate lyase for the CoA-like prosthetic group may arise from the substrate requirement of both subunit enzymes of the enzyme complex.     

Synthesis of pyruvate carboxylase from its apoenzyme and (+)-biotin in Bacillus stearothermophilus. Mechanism and control of the reaction

1. Acetyl-CoA acts as a positive allosteric effector in the formation of active pyruvate carboxylase from its apoenzyme, ATP and (+)-biotin which is catalysed by holoenzyme synthetase; this effect is counteracted by l-aspartate. 2. The Hill coefficients (apparent n values) were approximately 2 for acetyl-CoA and 4 for l-aspartate; the n value for each effector remained constant when the concentration of the other effector was varied. 3. Active pyruvate carboxylase was formed also when the apoenzyme was incubated with holoenzyme synthetase and synthetic biotinyl-5'-AMP; acetyl-CoA and l-aspartate affected this process as they did the overall reaction from (+)-biotin and ATP. 4. When hydroxylamine replaced the apoenzyme, holoenzyme synthetase catalysed the formation of biotinylhydroxamate from (+)-biotin and ATP. This reaction was not affected by the allosteric effectors. 5. The apoenzyme was protected against thermal denaturation by acetyl-CoA and, to a lesser degree, by l-aspartate. The holoenzyme synthetase was not markedly protected by these effectors. 6. It is concluded that the allosteric effectors act on the apoenzyme and not the synthetase.     

Isolation and characterization of active N-terminal truncated apo- and holoenzyme of mammalian liver tyrosine aminotransferase.

Limited proteolysis was used to probe the structure of the apo- and holoenzyme of rat liver tyrosine aminotransferase. Both were subjected to trypsinolysis and the major fragments were isolated and characterized. Trypsin cleaves the apoenzyme after residues Arg57, Lys64, and Lys71 and the holoenzyme after Arg37 and Lys38. The difference in the accessibility of the enzyme deprived or associated with pyridoxal 5'-phosphate reflects two distinct conformations. The activity, the affinity for the ligands and the thermostability of the purified truncated enzyme forms are similar to those of the native apo- and holoenzyme. A model for the domain structure of mammalian tyrosine aminotransferase and a mechanism for its rapid turnover are proposed.     

Distribution and transport of apo- and holocytochrome b5 in the endoplasmic reticulum of rat liver

The transport and distribution of apo- and holocytochrome $\text{b}{}_5$ was investigated with the aid of specific antibodies. The holoenzyme was found to be localized mainly in the rough and smooth endoplasmic reticulum and in the Golgi system but some precipitation could also be obtained in the outer mitochondrial membranes and in the peroxisomes. The apoenzyme, however, could only be detected in the endoplasmic reticulum-Golgi system, which also was shown to be the sole site for incorporation of the prosthetic heme moiety. Time-course studies revealed that the labeled enzyme appeared both as apoenzyme and as holoenzyme in the rough endoplasmic reticulum 10 min after in vivo injection of radioactive leucine and that further transport to the smooth endoplasmic reticulum occurred within 10 min. The subsequent transport to other organelles, however, required a somewhat longer time and peak radioactivity in outer mitochondrial membranes was not attained until after 40 min.     

Stability and Activation of Glutamate Apodecarboxylase from Pig Brain

The stability and activation of glutamate apodecarboxylase was studied with three forms of the enzyme from pig brain (referred to as the alpha, beta, and gamma forms). Apoenzyme was prepared by incubating the holoenzyme with aspartate followed by chromatography on Sephadex G-25. Apoenzyme was much less stable than holoenzyme to inactivation by heat (for beta-glutamate decarboxylase (beta-GAD) at 30 degrees C, t1/2 values of apo- and holoenzyme were 17 and greater than 100 min). ATP protected holoenzyme and apoenzyme against heat inactivation. The kinetics of reactivation of apoenzyme by pyridoxal-P was consistent with a two-step mechanism comprised of a rapid, reversible association of the cofactor with apoenzyme followed by a slow conversion of the complex to active holoenzyme. The reactivation rate constant (kr) and apparent dissociation constant (KD) for the binding of pyridoxal-P to apoenzyme differed substantially among the forms (for alpha-, beta-, and gamma-GAD, kr = 0.032, 0.17, and 0.27 min-1, and KD = 0.014, 0.018, and 0.04 microM). ATP was a strong competitive inhibitor of activation (Ki = 0.45, 0.18, and 0.39 microM for alpha-, beta-, and gamma-GAD). In contrast, Pi stimulated activation at 1-5 mM but inhibited at much higher concentrations. The results suggest that ATP is important in stabilizing the apoenzyme in brain and that ATP, Pi, and other compounds regulate its activation.     

The role of divalent cations in structure and function of murine adenosine deaminase.

For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo- and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo- or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N6- methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (Ki > or = 1000 microM) was noted for Ca2+, Fe2+, and Fe3+.     

Phosphopantethenylated Precursor Acyl Carrier Protein Is Imported into Spinach (Spinacia oleracea) Chloroplasts

Acyl carrier protein (ACP) is an essential cofactor of fatty acid synthase. In plants, ACP is synthesized in the cytosol as a larger precursor protein and then is imported into the plastid where it is processed to a smaller mature form. The active form of ACP uses a covalently linked 4[prime]-phosphopantetheine prosthetic group derived from coenzyme A to covalently bind the acyl intermediates during fatty acid synthesis. The prosthetic group is added to ACP by holoACP synthase. This enzyme activity is associated with both the plastidial subcellular fraction and the soluble, or cytoplasmic, fraction. To gain further insight into potential in vivo pathways for the synthesis and maturation of ACP, in this study we examined whether precursor holoACP can be imported by isolated spinach (Spinacia oleracea) chloroplasts. Precursor holoACP containing a [35S]phosphopantetheine prosthetic group was prepared, and the radiolabel was used to demonstrate import of the phosphopantethenylated protein into isolated chloroplasts. In addition, timed chloroplast import assays indicated that in vitro import of the phosphopantethenylated protein is at least as efficient as import of the precursor apoprotein. Evidence was also obtained for a low level turnover of the prosthetic group among endogenous plastidial ACPs when coenzyme A was supplied exogenously.     

Posttranslational Maturation of the Invasion Acyl Carrier Protein of Salmonella enterica Serovar Typhimurium Requires an Essential Phosphopantetheinyl Transferase of the Fatty Acid Biosynthesis Pathway

Salmonella pathogenicity island 1 (SPI-1) carries genes required for the formation of a type 3 secretion system, which is necessary for the invasion process of Salmonella. Among the proteins encoded by SPI-1 is IacP, a homolog of acyl carrier proteins. Acyl carrier proteins are mainly involved in fatty acid biosynthesis, and they require posttranslational maturation by addition of a 4′-phosphopantetheine prosthetic group to be functional. In this study, we analyzed IacP maturation in vivo. By performing matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) mass spectrometry analysis of intact purified proteins, we showed that IacP from Salmonella enterica serovar Typhimurium was matured by addition of 4′-phosphopantetheine to the conserved serine 38 residue. Therefore, we searched for the phosphopantetheinyl transferases in charge of IacP maturation. A bacterial two-hybrid approach revealed that IacP interacted with AcpS, an enzyme normally required for the maturation of the canonical acyl carrier protein (ACP), which is involved in fatty acid biosynthesis. The creation of a conditional acpS mutant then demonstrated that AcpS was necessary for the maturation of IacP. However, although IacP was similar to ACP and matured by using the same enzyme, IacP could not replace the essential function of ACP in fatty acid synthesis. Hence, the demonstration that IacP is matured by AcpS establishes a cross-connection between virulence and fatty acid biosynthesis pathways.     

Effect of the modification of carboxylic groups in protohemin IX on the properties of endoperoxide prostaglandin synthetase

The effect of protohemin IX and its modified analogs (monomethyl ester, dimethyl ester, as well as monoamides with Val-Phe-OCH3 or Leu-His-OCH3) has been examined on the activity of prostaglandin endoperoxide synthetase from sheep vesicular glands (PGH-synthetase, EC 1.14.99.1, isolated as apoenzyme). For holoenzymes having the above compounds as prosthetic groups, the dissociation constants, relative activities and the apparent inactivation constants in the course of the reaction have been determined. The effect of Tween 20 on the indicated parameters for holoenzymes with protohemin IX and its mono- and dimethyl esters has been studied. Modification of one of the two carboxylic groups of protohemin IX markedly increases the dissociation constant for the respective holoenzyme and virtually does not affect catalytic activity. Modification of both carboxylic groups of protohemin IX hinder the binding with the apoenzyme and strongly reduces the catalytic activity of the holoenzyme.