Identification by mutagenesis of conserved arginine and tryptophan residues in rat liver carnitine palmitoyltransferase I important for catalytic activity

Carnitine palmitoyltransferase I catalyzes the conversion of long-chain acyl-CoA to acylcarnitines in the presence of l-carnitine. To determine the role of the conserved arginine and tryptophan residues on catalytic activity in the liver isoform of carnitine palmitoyltransferase I (L-CPTI), we separately mutated five conserved arginines and two tryptophans to alanine. Substitution of arginine residues 388, 451, and 606 with alanine resulted in loss of 88, 82, and 93% of L-CPTI activity, respectively. Mutants R601A and R655A showed less than 2% of the wild type L-CPTI activity. A change of tryptophan 391 and 452 to alanine resulted in 50 and 93% loss in carnitine palmitoyltransferase activity, respectively. The mutations caused decreases in catalytic efficiency of 80-98%. The residual activity in the mutant L-CPTIs was sensitive to malonyl-CoA inhibition. Mutants R388A, R451A, R606A, W391A, and W452A had no effect on the K(m) values for carnitine or palmitoyl-CoA. However, these mutations decreased the V(max) values for both substrates by 10-40-fold, suggesting that the main effect of the mutations was to decrease the stability of the enzyme-substrate complex. We suggest that conserved arginine and tryptophan residues in L-CPTI contribute to the stabilization of the enzyme-substrate complex by charge neutralization and hydrophobic interactions. The predicted secondary structure of the 100-amino acid residue region of L-CPTI, containing arginines 388 and 451 and tryptophans 391 and 452, consists of four alpha-helices similar to the known three-dimensional structure of the acyl-CoA-binding protein. We predict that this 100-amino acid residue region constitutes the putative palmitoyl-CoA-binding site in L-CPTI.     

Identification by mutagenesis of conserved arginine and glutamate residues in the C-terminal domain of rat liver carnitine palmitoyltransferase I that are important for catalytic activity and malonyl-CoA sensitivity

Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long chain fatty acyl-CoAs to acylcarnitines in the presence of l-carnitine. To determine the role of the conserved glutamate residue, Glu-603, on catalysis and malonyl-CoA sensitivity, we separately changed the residue to alanine, histidine, glutamine, and aspartate. Substitution of Glu-603 with alanine or histidine resulted in complete loss of L-CPTI activity. A change of Glu-603 to glutamine caused a significant decrease in catalytic activity and malonyl-CoA sensitivity. Substitution of Glu-603 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild type enzyme, resulted in partial loss in CPTI activity and a 15-fold decrease in malonyl-CoA sensitivity. The mutant L-CPTI with a replacement of the conserved Arg-601 or Arg-606 with alanine also showed over 40-fold decrease in malonyl-CoA sensitivity, suggesting that these two conserved residues may be important for substrate and inhibitor binding. Since a conservative substitution of Glu-603 to aspartate or glutamine resulted in partial loss of activity and malonyl-CoA sensitivity, it further suggests that the negative charge and the longer side chain of glutamate are essential for catalysis and malonyl-CoA sensitivity. We predict that this region of L-CPTI spanning these conserved C-terminal residues may be the region of the protein involved in binding the CoA moiety of palmitoyl-CoA and malonyl-CoA and/or the putative low affinity acyl-CoA/malonyl-CoA binding site.     

A single amino acid change (substitution of the conserved Glu-590 with alanine) in the C-terminal domain of rat liver carnitine palmitoyltransferase I increases its malonyl-CoA sensitivity close to that observed with the muscle isoform of the enzyme

Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of l-carnitine. To determine the role of the highly conserved C-terminal glutamate residue, Glu-590, on catalysis and malonyl-CoA sensitivity, we separately changed the residue to alanine, lysine, glutamine, and aspartate. Substitution of Glu-590 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild-type enzyme, resulted in complete loss in the activity of the liver isoform of CPTI (L-CPTI). A change of Glu-590 to alanine, glutamine, and lysine caused a significant 9- to 16-fold increase in malonyl-CoA sensitivity but only a partial decrease in catalytic activity. Substitution of Glu-590 with neutral uncharged residues (alanine and glutamine) and/or a basic positively charged residue (lysine) significantly increased L-CPTI malonyl-CoA sensitivity to the level observed with the muscle isoform of the enzyme, suggesting the importance of neutral and/or positive charges in the switch of the kinetic properties of L-CPTI to the muscle isoform of CPTI. Since a conservative substitution of Glu-590 to aspartate but not glutamine resulted in complete loss in activity, we suggest that the longer side chain of glutamate is essential for catalysis and malonyl-CoA sensitivity. This is the first demonstration whereby a single residue mutation in the C-terminal region of the liver isoform of CPTI resulted in a change of its kinetic properties close to that observed with the muscle isoform of the enzyme and provides the rationale for the high malonyl-CoA sensitivity of muscle CPTI compared with the liver isoform of the enzyme.     

Ablation of the liver fatty acid binding protein gene decreases fatty acyl CoA binding capacity and alters fatty acyl CoA pool distribution in mouse liver

Although liver fatty acid binding protein (L-FABP) is known to bind not only long chain fatty acid (LCFA) but also long chain fatty acyl CoA (LCFA-CoA), the physiological significance of LCFA-CoA binding has been questioned and remains to be resolved. To address this issue, the effect of L-FABP gene ablation on liver cytosolic LCFA-CoA binding, LCFA-CoA pool size, LCFA-CoA esterification, and potential compensation by other intracellular LCFA-CoA binding proteins was examined. L-FABP gene ablation resulted not only in loss of L-FABP but also in concomitant upregulation of two other intracellular LCFA-CoA binding proteins, acyl CoA binding protein (ACBP) and sterol carrier protein-2 (SCP-2), by 45 and 80%, respectively. Nevertheless, the soluble fraction from livers of L-FABP (-/-) mice bound 95% less radioactive oleoyl-CoA than wild-type L-FABP (+/+) mice. The intracellular LCFA-CoA binding protein fraction (Fraction III) from wild-type L-FABP (+/+) mice, isolated by gel permeation chromatography of liver soluble proteins, exhibited one high-affinity binding and several low-affinity binding sites for cis-parinaroyl-CoA, a naturally occurring fluorescent LCFA-CoA. In contrast, high-affinity LCFA-CoA binding was absent from Fraction III of L-FABP (-/-) mice. While L-FABP gene ablation did not alter liver LCFA-CoA pool size, LCFA-CoA acyl chains of L-FABP (-/-) mouse livers were enriched 2.1-fold in C16:1 and decreased 1.9-fold in C20:0 fatty acids. Finally, L-FABP gene ablation selectively increased the amount of LCFAs esterified into liver phospholipid > cholesteryl ester, while concomitantly decreasing the amount of fatty acids esterified into triglycerides by 40%. In summary, these data with L-FABP (-/-) mice demonstrated for the first time that L-FABP is a physiologically significant contributor to determining liver cytosolic LCFA-CoA binding capacity, LCFA-CoA acyl chain distribution, and esterified fatty acid distribution.     

Holo-sterol carrier protein-2. (13)C NMR investigation of cholesterol and fatty acid binding sites

Although sterol carrier protein-2 (SCP-2) stimulates sterol transfer in vitro, almost nothing is known regarding the identity of the putative cholesterol binding site. Furthermore, the interrelationship(s) between this SCP-2 ligand binding site and the recently reported SCP-2 long chain fatty acid (LCFA) and long chain fatty acyl-CoA (LCFA-CoA) binding site(s) remains to be established. In the present work, two SCP-2 ligand binding sites were identified. First, both [4-(13)C]cholesterol and 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol) binding assays were consistent with a single cholesterol binding site in SCP-2. This ligand binding site had high affinity for NBD-cholesterol, K(d) = 4.15 +/- 0.71 nM. (13)C NMR-labeled ligand competition studies demonstrated that the SCP-2 high affinity cholesterol binding site also bound LCFA or LCFA-CoA. However, only the LCFA-CoA was able to effectively displace the SCP-2-bound [4-(13)C]cholesterol. Thus, the ligand affinities at this SCP-2 binding site were in the relative order cholesterol = LCFA-CoA > LCFA. Second, (13)C NMR studies demonstrated the presence of another ligand binding site on SCP-2 that bound either LCFA or LCFA-CoA but not cholesterol. Photon correlation spectroscopy was consistent with SCP-2 being monomeric in both liganded and unliganded states. In summary, both (13)C NMR and fluorescence techniques demonstrated for the first time that SCP-2 had a single high affinity binding site that bound cholesterol, LCFA, or LCFA-CoA. Furthermore, results with (13)C NMR supported the presence of a second SCP-2 ligand binding site that bound either LCFA or LCFA-CoA but not cholesterol. These data contribute to our understanding of a role for SCP-2 in both cellular cholesterol and LCFA metabolism.     

Role of regulatory F-domain in hepatocyte nuclear factor-4alpha ligand specificity

The F-domain of rat HNF-4alpha1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4alpha. (i) Fluorescent binding assays indicate that wild-type, full-length HNF-4alpha (amino acids 1-455) has high affinity (Kd=0.06-12 nm) for long chain fatty acyl-CoAs (LCFA-CoA) and low affinity (Kd=58-296 nm) for unesterified long chain fatty acids (LCFAs). LCFA-CoA binding was due to close molecular interaction as shown by fluorescence resonance energy transfer (FRET) from full-length HNF-4alpha tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor), which yielded an intermolecular distance of 33 A, although no FRET to cis-parinaric acid was detected. (ii) Deleting the N-terminal A-D-domains, comprising the AF1 and DNA binding functions, only slightly affected affinities for LCFA-CoAs (Kd=0.9-4 nm) and LCFAs (Kd=93-581 nm). (iii) Further deletion of the F-domain robustly reduced affinities for LCFA-CoA and reversed ligand specificity (i.e. high affinity for LCFAs (Kd=1.5-32 nm) and low affinity for LCFA-CoAs (Kd=54-302 nm)). No FRET from HNF-4alpha-E (amino acids 132-370) tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor) was detected, whereas an intermolecular distance of 28 A was calculated from FRET between HNF-4alpha-E and cis-parinaric acid. (iv) Circular dichroism showed that LCFA-CoA, but not LCFA, altered the secondary structure of HNF-4alpha only when the F-domain was present. (v) cis-Parinaric acid bound to HNF-4alpha with intact F-domain was readily displaceable by S-hexadecyl-CoA, a nonhydrolyzable thioether analogue of LCFA-CoAs. Truncation of the F-domain significantly decreased cis-parinaric acid displacement. Hence, the C-terminal F-domain of HNF-4alpha regulated ligand affinity, ligand specificity, and ligand-induced conformational change of HNF-4alpha. Thus, characteristics of F-domain-truncated mutants may not reflect the properties of full-length HNF-4alpha.     

Pig liver carnitine palmitoyltransferase I, with low Km for carnitine and high sensitivity to malonyl-CoA inhibition, is a natural chimera of rat liver and muscle enzymes

The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of fatty acids. The genes for the two isoforms of CPTI-liver (L-CPTI) and muscle (M-CPTI) have been cloned and expressed, and the genes encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition. Pig L-CPTI encodes for a 772 amino acid protein that shares 86 and 62% identity, respectively, with rat L- and M-CPTI. When expressed in Pichia pastoris, the pig L-CPTI enzyme shows kinetic characteristics (carnitine, K(m) = 126 microM; palmitoyl-CoA, K(m) = 35 microM) similar to human or rat L-CPTI. However, the pig enzyme, unlike the rat liver enzyme, shows a much higher sensitivity to malonyl-CoA inhibition (IC(50) = 141 nM) that is characteristic of human or rat M-CPTI enzymes. Therefore, pig L-CPTI behaves like a natural chimera of the L- and M-CPTI isotypes, which makes it a useful model to study the structure--function relationships of the CPTI enzymes.     

Acyl-coenzyme A binding protein expression alters liver fatty acyl-coenzyme A metabolism

Although studies in vitro and in yeast suggest that acyl-CoA binding protein ACBP may modulate long-chain fatty acyl-CoA (LCFA-CoA) distribution, its physiological function in mammals is unresolved. To address this issue, the effect of ACBP on liver LCFA-CoA pool size, acyl chain composition, distribution, and transacylation into more complex lipids was examined in transgenic mice expressing a higher level of ACBP. While ACBP transgenic mice did not exhibit altered body or liver weight, liver LCFA-CoA pool size increased by 69%, preferentially in saturated and polyunsaturated, but not monounsaturated, LCFA-CoAs. Intracellular LCFA-CoA distribution was also altered such that the ratio of LCFA-CoA content in (membranes, organelles)/cytosol increased 2.7-fold, especially in microsomes but not mitochondria. The increased distribution of specific LCFA-CoAs to the membrane/organelle and microsomal fractions followed the same order as the relative LCFA-CoA binding affinity exhibited by murine recombinant ACBP: saturated > monounsaturated > polyunsaturated C14-C22 LCFA-CoAs. Consistent with the altered microsomal LCFA-CoA level and distribution, enzymatic activity of liver microsomal glycerol-3-phosphate acyltransferase (GPAT) increased 4-fold, liver mass of phospholipid and triacylglyceride increased nearly 2-fold, and relative content of monounsaturated C18:1 fatty acid increased 44% in liver phospholipids. These effects were not due to the ACBP transgene altering the protein levels of liver microsomal acyltransferase enzymes such as GPAT, lysophosphatidic acid acyltransferase (LAT), or acyl-CoA cholesterol acyltransferase 2 (ACAT-2). Thus, these data show for the first time in a physiological context that ACBP expression may play a role in LCFA-CoA metabolism.     

Pig muscle carnitine palmitoyltransferase I (CPTI beta), with low Km for carnitine and low sensitivity to malonyl-CoA inhibition, has kinetic characteristics similar to those of the rat liver (CPTI alpha) enzyme

The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of long-chain fatty acids. There are two well-characterized isotypes of CPTI: CPTIalpha (also known as L-CPTI) and CPTIbeta (also known as M-CPTI) that in human and rat encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition. Kinetic hallmarks of the CPTIalpha are high affinity for carnitine and low sensitivity to malonyl-CoA inhibition, while the opposite characteristics, low affinity for carnitine and high sensitivity to malonyl-CoA, are intrinsic to the CPTIbeta isotype. We have isolated the pig CPTIbeta cDNA which encodes for a protein of 772 amino acids that shares extensive sequence identity with human (88%), rat (85%), and mouse (86%) CPTIbeta, while the degree of homology with the CPTIalpha from human (61%), rat (62%), and mouse (60%) is much lower. However, when expressed in the yeast Pichia pastoris, pig CPTIbeta shows kinetic characteristics similar to those of the CPTIalpha isotype. Thus, the pig CPTIbeta, unlike the corresponding human or rat enzyme, has a high affinity for carnitine (K(m) = 197 microM) and low sensitive to malonyl-CoA inhibition (IC(50) = 906 nM). Therefore, the recombinant pig CPTIbeta has unique kinetic characteristics, which makes it a useful model to study the structure-function relationship of the CPTI enzymes.     

Biochemical characterisation of a hydrophobic ligand binding protein from the tapeworm Hymenolepis diminuta

The cestode Hymenolepis diminuta contains an abundant, cytoplasmic, hydrophobic ligand, binding protein (H-HLBP). Studies with polarity sensitive probes suggest a single hydrophobic binding site, the results also indicate that the single tryptophan in the molecule (Trp41) is involved in ligand binding. Of the possible physiological ligands tested, only haematin and retinoids (retinol and retinoic acid) show appreciable binding in addition to fatty acids. H-HLBP also binds a range of anthelmintics, again with K(D) values in the nM range. The interaction of anthelmintics with hydrophobic binding proteins may be important in determining drug specificity and site of action and could have a role in the development of drug resistance.     

Analysis of tryptophan‐rich region in Clostridium septicum alpha‐toxin involved with binding to glycosylphosphatidylinositol‐anchored proteins

Clostridium septicum alpha‐toxin has a unique tryptophan‐rich region (³⁰²NGYSEWDWKWV³¹²) that consists of 11 amino acid residues near the C‐terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan‐rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)‐anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI‐anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan‐rich region plays an important role in the cytotoxic mechanism of alpha‐toxin, especially in the binding to GPI‐anchored proteins as cell receptors.     

Parinaric acids as probes of binding domains in neutrophil elastase

Human neutrophil elastase has an extended hydrophobic substrate binding site which serves as a target for a number of hydrophobic inhibitors. We show here that the parinaric acids, fluorescent-conjugated tetraenoic fatty acids of plant origin, are inhibitors of neutrophil elastase. cis-Parinaric acid (cis-PA) interacts with the enzyme in two inhibitory modes. The high affinity interaction (Ki = 55 +/- 6 nM) results in partial noncompetitive inhibition of amidolytic activity, with 82% residual activity. A lower affinity interaction with cis-PA (Ki = 4 +/- 1 microM) results in competitive inhibition. trans-PA also acts as a high affinity partial noncompetitive inhibitor of elastase with a Ki equal to that for cis-PA but has no low affinity competitive inhibitory action. The endogenous fluorescence from the 3 tryptophan residues in elastase is partially quenched on binding cis- or trans-PA. Dependence of quenching of tryptophan fluorescence on PA concentration is consistent with binding to a single site with an apparent Kd of 26 +/- 3 nM, which may be equivalent to the high affinity partial noncompetitive inhibitory binding mode. Analysis of quenching according to the modified Forster theory of energy transfer developed by Snyder and Freire (Snyder, B., and Freire, E. (1982) Biophys. J. 40, 137-148) leads to an estimate of apparent closest indole-PA distance of 13 +/- 3 A. Fluorescence of either cis- or trans-PA is apparently unperturbed upon binding in the high affinity mode to elastase, but at micromolar cis-PA concentrations, binding to elastase results in a blue shift and 20% increase in intensity of PA emission, suggesting that the lower affinity competitive inhibitory binding mode of binding to elastase provides a hydrophobic environment for cis-PA.     

Functional roles of Tryptophan residues in diketoreductase from Acinetobacter baylyi

Diketoreductase (DKR) from Acinetobacter baylyi contains two tryptophan residues at positions 149 and 222. Trp-149 and Trp-222 are located along the entry path of substrate into active site and at the dimer interface of DKR, respectively. Single and double substitutions of these positions were generated to probe the roles of tryptophan residues. After replacing Trp with Ala and Phe, biochemical and biophysical characteristics of the mutants were thoroughly investigated. Enzyme activity and substrate binding affinity of W149A and W149F were remarkably decreased, suggesting that Trp-149 regulates the position of substrate at the binding site. Meanwhile, enzyme activity of W222F was increased by 1.7-fold while W222A was completely inactive. In addition to lower thermostability of Trp-222 mutants, molecular modeling of the mutants revealed that Trp-222 is vital to protein folding and dimerization of the enzyme. [BMB Reports 2012; 45(8): 452-457].     

Cellular uptake and intracellular trafficking of long chain fatty acids.

While aspects of cellular fatty acid uptake have been studied as early as 50 years ago, recent developments in this rapidly evolving field have yielded new functional insights on the individual mechanistic steps in this process. The extremely low aqueous solubility of long chain fatty acids (LCFA) together with the very high affinity of serum albumin and cytoplasmic fatty acid binding proteins for LCFA have challenged the limits of technology in resolving the individual steps of this process. To date no single mechanism alone accounts for regulation of cellular LCFA uptake. Key regulatory points in cellular uptake of LCFA include: the aqueous solubility of the LCFA; the driving force(s) for LCFA entry into the cell membrane; the relative roles of diffusional and protein mediated LCFA translocation across the plasma membrane; cytoplasmic LCFA binding protein-mediated uptake and/or intracellular diffusion; the activity of LCFA-CoA synthetase; and cytoplasmic protein mediated targeting of LCFA or LCFA-CoAs toward specific metabolic pathways. The emerging picture is that the cell has multiple, overlapping mechanisms that assure adequate uptake and directed intracellular movement of LCFA required for maintenance of physiological functions. The upcoming challenge is to take advantage of new advances in this field to elucidate the differential interactions between these pathways in intact cells and in tissues.     

Rhodopsin activation exposes a key hydrophobic binding site for the transducin alpha-subunit C terminus

Conformational changes enable the photoreceptor rhodopsin to couple with and activate the G-protein transducin. Here we demonstrate a key interaction between these proteins occurs between the C terminus of the transducin alpha-subunit (G(Talpha)) and a hydrophobic cleft in the rhodopsin cytoplasmic face exposed during receptor activation. We mapped this interaction by labeling rhodopsin mutants with the fluorescent probe bimane and then assessed how binding of a peptide analogue of the G(Talpha) C terminus (containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the G(Talpha) C-terminal tail binds to the inner face of helix 6 in a retinal-linked manner. Further, we find that a "hydrophobic patch" comprising key residues in the exposed cleft is required for transducin binding/activation because it enhances the binding affinity for the G(Talpha) C-terminal tail, contributing up to 3 kcal/mol for this interaction. We speculate the hydrophobic interactions identified here may be important in other GPCR signaling systems, and our Trp/bimane fluorescence methodology may be generally useful for mapping sites of protein-protein interaction.     

The extreme C terminus of rat liver carnitine palmitoyltransferase I is not involved in malonyl-CoA sensitivity but in initial protein folding

We previously reported that the N-terminal domain (1-147 residues) of rat liver carnitine palmitoyltransferase I (L-CPTI) was essential for import into the outer mitochondrial membrane and for maintenance of a malonyl-CoA-sensitive conformation. Malonyl-CoA binding experiments using mitochondria of Saccharomyces cerevisiae strains expressing wild-type L-CPTI or previously constructed chimeric CPTs (Cohen, I., Kohl, C., McGarry, J.D., Girard, J., and Prip-Buus, C. (1998) J. Biol. Chem. 273, 29896-29904) indicated that the N-terminal domain was unable, independently of the C-terminal domain, to bind malonyl-CoA with a high affinity, suggesting that the modulation of malonyl-CoA sensitivity occurred through N/C intramolecular interactions. To assess the role of the C terminus in malonyl-CoA sensitivity, a series of C-terminal deletion mutants was generated. The kinetic properties of Delta772-773 and Delta767-773 deletion mutants were similar to those of L-CPTI, indicating that the last two highly conserved Lys residues in all known L-CPTI species were not functionally essential. By contrast, Delta743-773 deletion mutant was totally inactive and unfolded, as shown by its sensitivity to trypsin proteolysis. Because the C terminus of the native folded L-CPTI could be cleaved by trypsin without inducing protein unfolding, we concluded that the last 31 C-terminal residues constitute a secondary structural determinant essential for the initial protein folding of L-CPTI.     

A single amino acid change (substitution of glutamate 3 with alanine) in the N-terminal region of rat liver carnitine palmitoyltransferase I abolishes malonyl-CoA inhibition and high affinity binding

We have recently shown by deletion mutation analysis that the conserved first 18 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) are essential for malonyl-CoA inhibition and binding (Shi, J., Zhu, H., Arvidson, D. N. , Cregg, J. M., and Woldegiorgis, G. (1998) Biochemistry 37, 11033-11038). To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we constructed two more deletion mutants, Delta12 and Delta6, and three substitution mutations within the conserved first six amino acid residues. Mutant L-CPTI, lacking either the first six N-terminal amino acid residues or with a change of glutamic acid 3 to alanine, was expressed at steady-state levels similar to wild type and had near wild type catalytic activity. However, malonyl-CoA inhibition of these mutant enzymes was reduced 100-fold, and high affinity malonyl-CoA binding was lost. A mutant L-CPTI with a change of histidine 5 to alanine caused only partial loss of malonyl-CoA inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to alanine had wild type properties. These results demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl-CoA binding and inhibition of L-CPTI by malonyl-CoA but are not required for catalysis.     

Serine to cysteine mutations in trp repressor protein alter tryptophan and operator binding

The tryptophan repressor regulates expression of the aroH, trpEDCBA, and trpR operons in Escherichia coli. The protein contains no cysteine residues, and the presence of this reactive side chain would allow introduction of spectral probes to monitor binding reactions. Three mutant trp aporepressors, each with a point mutation from serine to cysteine, were produced at positions 67, 86, and 88 by oligonucleotide-directed site-specific mutagenesis. This single conservative substitution affected both tryptophan and operator DNA affinities in all three purified proteins. Cysteine substitution for serine at position 67 decreased tryptophan binding by approximately 6-fold and the operator DNA affinity by approximately 50-fold. The proximity of this amino acid to Gln-68 which is involved in binding to operator DNA (Otwinowski, Z., Schevitz, R. W., Zhang, R.-G., Lawson, C. L., Joachimiak, A., Marmorstein, R. Q., Luisi, B. F., and Sigler, P. B. (1988) Nature 335, 321-329) may account for this effect. Substitution at position 86 diminished tryptophan binding by approximately 4-fold and operator DNA binding by approximately 130-fold. The participation of Ser-86 in the hydrogen bond network required for operator binding (Otwinowski, Z., Schevitz, R. W., Zhang, R.-G., Lawson, C. L., Joachimiak, A., Marmorstein, R. Q., Luisi, B. F., and Sigler, P. B. (1988) Nature 335, 321-329) presumably accounts for the DNA binding effects. The diminished corepressor activity in these two mutants may derive from distortions of the binding region, as the tryptophan and DNA binding sites are intimately related. The mutation at position 88 altered tryptophan binding the most of the three mutants (approximately 18-fold) and operator binding least (approximately 12-fold). Ser-88 forms a hydrogen bond with the amino group of bound tryptophan (Schevitz, R. W., Otwinowski, Z., Joachimiak, A., Lawson, C. L., and Sigler, P. B. (1985) Nature 317, 782-786), and alteration of the geometry of the side chain would be anticipated to perturb the topology of the binding site. The diminished operator affinity may derive from improper alignment of the tryptophan ligand, crucial for high affinity operator binding (Otwinowski, Z., Schevitz, R. W., Zhang, R.-G., Lawson, C. L., Joachimiak, A., Marmorstein, R. Q., Luisi, B. F., and Sigler, P. B. (1988) Nature 335, 321-329).(ABSTRACT TRUNCATED AT 400 WORDS)     

Islet amyloid polypeptide (IAPP) competes for two binding sites of CGRP.

Two distinct binding sites for [125I]human calcitonin gene-related peptide (hCGRP) were found in rat brain, skeletal muscle, and liver. Each tissue had a high affinity site with an average Kd of 46 pM and a low affinity site with an average Kd of 22 nM. Islet amyloid polypeptide (IAPP), which has N- and C-terminal sequence homology to CGRP and is produced by islet beta-cells, bound to both sites but had a potency closer to that of CGRP at the low affinity binding site. A C-terminal fragment of IAPP competed for [125I]hCGRP binding at the low affinity site with potency comparable to that of hIAPP. No specific binding to membrane preparations was found in experiments using [125I]rIAPP, which was iodinated at the C-terminal tyrosyl residue. These results suggest that some of the previously reported biological effects occurring at nM or microM concentrations of IAPP may be mediated by IAPP binding to low affinity CGRP receptors. This study further indicates that the C-terminal region of IAPP is important for binding to low affinity CGRP receptors, and suggests that C-terminal fragments of IAPP may be of biological importance.     

Binding properties of Echinococcus granulosus fatty acid binding protein.

EgFABP1 is a developmentally regulated intracellular fatty acid binding protein characterized in the larval stage of parasitic platyhelminth Echinococcus granulosus. It is structurally related to the heart group of fatty acid binding proteins (H-FABPs). Binding properties and ligand affinity of recombinant EgFABP1 were determined by fluorescence spectroscopy using cis- and trans-parinaric acid. Two binding sites for cis- and trans-parinaric acid were found (K(d(1)) 24+/-4 nM, K(d(2)) 510+/-60 nM for cis-parinaric acid and K(d(1)) 32+/-4 nM, K(d(2)) 364+/-75 nM for trans-parinaric). A putative third site for both fatty acids is discussed. Binding preferences were determined using displacement assays. Arachidonic and oleic acids presented the highest displacement percentages for EgFABP1. The Echinococcus FABP is the unique member of the H-FABP group able to bind two long chain fatty acid molecules with high affinity. Structure-function relationships and putative roles for EgFABP1 in E. granulosus metabolism are discussed.