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W1963817811 abstract "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, Δ12 and Δ6, 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. 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, Δ12 and Δ6, 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. carnitine palmitoyltransferase I rat liver isoform of CPTI heart/skeletal isoform of CPTI Carnitine palmitoyltransferase I (CPTI)1 catalyzes the conversion of long chain acyl-CoA to acylcarnitines in the presence ofl-carnitine, the first reaction in the transport of long chain fatty acids from the cytoplasm to the mitochondria, a rate-limiting step in β-oxidation (1Bieber L.L. Annu. Rev. Biochem. 1988; 57: 261-283Crossref PubMed Scopus (667) Google Scholar, 2McGarry J.D. Woeltje K.F. Kuwajima M. Foster D.W. Diabetes Metab. Rev. 1989; 5: 271-284Crossref PubMed Scopus (287) Google Scholar). Mammalian tissues express two isoforms of CPTI, a liver isoform (L-CPTI) and a heart/skeletal muscle isoform (M-CPTI), that are 62% identical in amino acid sequence (GenBankTM accession number U62317; Refs. 3Weis B.C. Esser V. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 18712-18715Abstract Full Text PDF PubMed Google Scholar, 4Weis B.C. Cowan A.T. Brown N. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 26443-26448Abstract Full Text PDF PubMed Google Scholar, 5Brown N.F. Weis B.C. Husti J.E. Foster D.W. McGarry J.D. J. Biol. Chem. 1995; 270: 8952-8957Crossref PubMed Scopus (136) Google Scholar, 6Zhu H. Shi J. de Vries Y. Arvidson D.N. Cregg J.M. Woldegiorgis G. Arch. Biochem. Biophys. 1997; 347: 53-61Crossref PubMed Scopus (51) Google Scholar, 7Yamazaki N. Shinhara Y. Shima A. Terada H. FEBS Lett. 1995; 363: 41-45Crossref PubMed Scopus (110) Google Scholar and 9Yamazaki N. Shinhara Y. Shima A. Yamanaka Y. Terada H. Biochim. Biophys. Acta. 1996; 1307: 157-161Crossref PubMed Scopus (102) Google Scholar). As an enzyme that catalyzes the first rate-limiting step in fatty acid oxidation, CPTI is regulated by its physiological inhibitor malonyl-CoA (1Bieber L.L. Annu. Rev. Biochem. 1988; 57: 261-283Crossref PubMed Scopus (667) Google Scholar, 2McGarry J.D. Woeltje K.F. Kuwajima M. Foster D.W. Diabetes Metab. Rev. 1989; 5: 271-284Crossref PubMed Scopus (287) Google Scholar), the first intermediate in fatty acid synthesis, suggesting coordinated control of fatty acid oxidation and synthesis. Understanding the molecular mechanism of the regulation of CPTI by malonyl-CoA is important in the design of drugs for control of excessive fatty acid oxidation in diabetes mellitus (10Prentki M. Corkey B.E. Diabetes. 1996; 45: 273-283Crossref PubMed Scopus (0) Google Scholar) and in myocardial ischemia where accumulation of acylcarnitines has been associated with arrhythmias (11Corr P.B. Yamada K.A. Herz. 1995; 20: 156-168PubMed Google Scholar).We developed a novel high level expression system for rat L-CPTI and human heart M-CPTI in the yeast Pichia pastoris, an organism devoid of endogenous CPT activity (6Zhu H. Shi J. de Vries Y. Arvidson D.N. Cregg J.M. Woldegiorgis G. Arch. Biochem. Biophys. 1997; 347: 53-61Crossref PubMed Scopus (51) Google Scholar, 12de Vries Y. Arvidson D.N. Waterham H.R. Cregg J.M. Woldegiorgis G. Biochemistry. 1997; 36: 5285-5292Crossref PubMed Scopus (52) Google Scholar, 13Zhu H. Shi J. Cregg J.M. Woldegiorgis G. Biochem. Biophys. Res. Commun. 1997; 239: 498-502Crossref PubMed Scopus (29) Google Scholar). Using this system, we demonstrated conclusively that L-CPTI and M-CPTI are active, distinct, malonyl-CoA-sensitive CPTIs that are reversibly inactivated by detergents. We recently showed that deletion of the conserved first 18 N-terminal amino acid residues of rat L-CPTI abolishes malonyl-CoA inhibition and high affinity malonyl-CoA binding (14Shi J. Zhu H. Arvidson D.N. Cregg J.M. Woldegiorgis G. Biochemistry. 1998; 37: 11033-11038Crossref PubMed Scopus (44) Google Scholar). In this study, we have constructed and characterized rat L-CPTI deletion mutants of the first 12 and 6 N-terminal amino acid residues. To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we also constructed three substitution mutations within the conserved first 6 N-terminal amino acid residues (Glu3 → Ala, His5 → Ala, and Gln6 → Ala).DISCUSSIONTo determine the role of the first 130 N-terminal amino acid residues of rat L-CPTI in malonyl-CoA sensitivity and binding, we previously constructed a series of deletion mutants and demonstrated that a mutant lacking the first conserved 18 N-terminal amino acid residues had activity and kinetic properties similar to those of wild type L-CPTI but had completely lost malonyl-CoA sensitivity and high affinity binding (14Shi J. Zhu H. Arvidson D.N. Cregg J.M. Woldegiorgis G. Biochemistry. 1998; 37: 11033-11038Crossref PubMed Scopus (44) Google Scholar). Based on these previous studies (14Shi J. Zhu H. Arvidson D.N. Cregg J.M. Woldegiorgis G. Biochemistry. 1998; 37: 11033-11038Crossref PubMed Scopus (44) Google Scholar), we report here on deletion mutations of the conserved first 12 and 6 N-terminal residues of L-CPTI. Like Δ18, Δ12 and Δ6 had 60–70% of the wild type activity and showed loss of both malonyl-CoA sensitivity and high affinity malonyl-CoA binding, indicating that residue(s) essential for malonyl-CoA binding and sensitivity reside within the conserved first 6 N-terminal amino acids. Of these conserved first 6 N-terminal amino acids, including the start codon Met, residues 2 and 4 are Ala, residue 3 is Glu, residue 5 is His, and residue 6 is Gln. Therefore, we constructed mutants with substitutions of Glu3 with Ala, His5 with Ala, and Gln6 with Ala of L-CPTI.The mutant L-CPTI with a replacement of Glu3 with Ala had a phenotype similar to that of the N-terminal deletion mutants. The mutation resulted in complete loss of malonyl-CoA sensitivity and high affinity malonyl-CoA binding and a decrease in the low affinity malonyl-CoA binding. In contrast, substitution of Glu3 with Ala did not have a significant effect on the kinetic properties of the enzyme, because there was no change in the K m value for palmitoyl-CoA and only a slight increase in theK m value for carnitine. The 29–40% loss in catalytic activity observed with the deletion and point mutants compared with the wild type could be due to a reduction in the expression level or lack of interaction of the N-terminal domain with the catalytic domain as a result of the N-terminal mutations. A protein of the expected size (88 kDa) was detected in the mitochondria of the Glu3 → Ala mutant strain on immunoblotting with L-CPTI specific antibodies. These results demonstrate clearly that Glu3 in the wild type L-CPTI is essential for malonyl-CoA inhibition and binding but not for catalysis, because the kinetic properties of the mutant enzyme are virtually indistinguishable from those of the wild type. This is the first report to demonstrate the critical role of Glu3 residue of L-CPTI for malonyl-CoA sensitivity and binding.The high affinity site (K D1,B max1) for binding of malonyl-CoA to L-CPTI was completely abolished in the Glu3 → Ala, Δ6, and Δ18 mutants, suggesting that the >100-fold decrease in malonyl-CoA sensitivity observed in these mutants was due to the loss of the high affinity binding entity of the enzyme. Although low affinity malonyl-CoA binding was weakened, there was no change in theB max2 value between wild type L-CPTI and mutants Glu3 → Ala, Δ6, and Δ18, suggesting that the residual malonyl-CoA sensitivity observed in the mutants was due to the low affinity malonyl-CoA-binding entity of the enzyme. The results of this study provide strong evidence implicating Glu3 as one of the residues involved in high affinity malonyl-CoA binding. We hypothesize that the Glu3 → Ala substitution may disrupt a hydrogen bonding network or a salt bridge, perhaps to a residue near the active site of CPTI. As the high affinity site is abolished and binding to the low affinity site is weakened, the two sites may partially overlap. Alternatively, the possible loss of a salt bridge may weaken K D2 indirectly.Replacement of His5 with Ala had a much less drastic effect on the IC50 for malonyl-CoA inhibition of L-CPTI but severely diminished both high and low affinity malonyl-CoA binding. TheB max1 for this mutant showed a slight increase, but B max2 showed a significant decrease compared with the wild type value, suggesting that the 10-fold lower IC50 for malonyl-CoA inhibition observed with this mutant, compared with mutants Glu3 → Ala, Δ6, and Δ18, may be due to a slight increase in abundance of the high affinity binding entity with a lowered (100-fold) affinity for malonyl-CoA. The decrease in low affinity malonyl-CoA binding observed for the His5→ Ala mutant (∼15-fold increase in K D2) may be due, in part, to the decreased abundance of the low affinity binding entity of the enzyme (∼3-fold decrease in B max2). Because mutation of His5 → Ala reduced the malonyl-CoA sensitivity and binding, L-CPTI may be affected by pH. A pH-induced shift in malonyl-CoA sensitivity has been reported for CPTI (25Stephens T.W. Cook G.A. Harris R.A. Biochem. J. 1983; 212: 521-524Crossref PubMed Scopus (32) Google Scholar, 26Mills S.E. Foster D.W. McGarry J.D. Biochem. J. 1984; 219: 601-608Crossref PubMed Scopus (50) Google Scholar).Our data clearly demonstrate that there are two classes of malonyl-CoA-binding sites in L-CPTI, namely, a high affinity and a low affinity binding site, similar to earlier studies in isolated rat liver and heart mitochondria (26Mills S.E. Foster D.W. McGarry J.D. Biochem. J. 1984; 219: 601-608Crossref PubMed Scopus (50) Google Scholar, 27Bird M.I. Saggerson E.D. Biochem. J. 1984; 222: 639-647Crossref PubMed Scopus (42) Google Scholar). A previous attempt to express a mutant L-CPTI that lacked the first 82 N-terminal residues was described by Brown et al. (28Brown N.F. Esser V. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 26438-26442Abstract Full Text PDF PubMed Google Scholar), but results were inconclusive due to extremely low expression levels (12de Vries Y. Arvidson D.N. Waterham H.R. Cregg J.M. Woldegiorgis G. Biochemistry. 1997; 36: 5285-5292Crossref PubMed Scopus (52) Google Scholar). The residual malonyl-CoA sensitivity shown by the deletion mutants is similar to that observed with yeast-expressed CPTII (12de Vries Y. Arvidson D.N. Waterham H.R. Cregg J.M. Woldegiorgis G. Biochemistry. 1997; 36: 5285-5292Crossref PubMed Scopus (52) Google Scholar), suggesting that for these mutants malonyl-CoA may inhibit via direct interaction with the active site. Additional studies are needed to determine whether the active site acts as a low affinity malonyl-CoA-binding site, but our data suggest that there may be some overlap between the malonyl-CoA and palmitoyl-CoA binding sites. In the absence of malonyl-CoA, free CoA (50 μm) and acetyl-CoA (500 μm) inhibited the activities of both the wild type and the Glu3 → Ala mutant L-CPTI by 50%. 2J. Shi, H. Zhu, D. N. Arvidson, and G. Woldegiorgis, unpublished observation.Because a total loss of the high affinity malonyl-CoA binding site was observed in the Glu3 → Ala mutant, the results suggest that CoA and acetyl-CoA inhibit by binding to the active site or the low affinity malonyl-CoA-binding site. At high concentrations, both CoA and the substrate palmitoyl-CoA reduce the inhibition of L-CPTI by malonyl-CoA (18Bremer J. Woldegiorgis G. Schalinske K. Shrago E. Biochim. Biophys. Acta. 1985; 833: 9-16Crossref PubMed Scopus (67) Google Scholar, 29Cook G.A. Mynatt R.L. Kashfi K. J. Biol. Chem. 1994; 269: 8803-8807Abstract Full Text PDF PubMed Google Scholar), suggesting partial overlap between the malonyl-CoA and the substrate binding sites.Based on limited proteolysis studies of intact and outer membrane rat liver mitochondria, a model for the membrane topology of L-CPTI has been proposed that predicts exposure of 90% of L-CPTI, including N and C termini domains crucial for activity and malonyl-CoA sensitivity of the enzyme on the cytosolic side of the outer mitochondrial membrane (30Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (122) Google Scholar). A more recent detailed deletion mutation analysis study of the 129 N-terminal amino acid residues of the yeast-expressed L-CPTI from our laboratory clearly demonstrated that residues critical for malonyl-CoA inhibition and binding of L-CPTI are located within the conserved first 18 N-terminal amino acid residues of the enzyme (14Shi J. Zhu H. Arvidson D.N. Cregg J.M. Woldegiorgis G. Biochemistry. 1998; 37: 11033-11038Crossref PubMed Scopus (44) Google Scholar). In this study, we demonstrate that glutamic acid residue 3 and histidine 5 are essential for malonyl-CoA binding and inhibition.Limited proteolysis of intact and outer membrane preparations of rat liver mitochondria result in a marked loss in L-CPTI activity and malonyl-CoA sensitivity (30Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (122) Google Scholar, 31Kashfi K. Mynatt R.L. Cook G.A. Biochim. Biophys. Acta. 1994; 1212: 245-252Crossref PubMed Scopus (31) Google Scholar), accompanied by the cleavage of the extreme N terminus (<1 kDa) of L-CPTI (30Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (122) Google Scholar). Mitochondria isolated from fasted and diabetic rat livers, metabolic conditions with increased fatty acid oxidation, exhibit increased L-CPTI activity and decreased malonyl-CoA sensitivity (32Cook G.A. Gamble M.S. J. Biol. Chem. 1987; 262: 2050-2055Abstract Full Text PDF PubMed Google Scholar). Furthermore, insulin reverses the effects of diabetes on L-CPTI activity and malonyl-CoA sensitivity (32Cook G.A. Gamble M.S. J. Biol. Chem. 1987; 262: 2050-2055Abstract Full Text PDF PubMed Google Scholar). Thus, fasting and diabetes, metabolic conditions that enhance protein degradation, reduce the sensitivity of CPTI to malonyl-CoA inhibition (32Cook G.A. Gamble M.S. J. Biol. Chem. 1987; 262: 2050-2055Abstract Full Text PDF PubMed Google Scholar, 33Park E.A. Mynatt R.L. Cook G.A. Kashfi K. Biochem. J. 1995; 310: 853-858Crossref PubMed Scopus (80) Google Scholar, 34Kashfi K. Cagen L. Cook G.A. Lipids. 1995; 30: 383-388Crossref PubMed Scopus (5) Google Scholar). The L-CPTI gene in INS-1 cells may be an early response gene like c-fos (35Assimacopoulos-Jeannet F. Thumelin S. Roche E. Esser V. McGarry J.D. Prentki M. J. Biol. Chem. 1997; 272: 1659-1664Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), suggesting that the enzyme may be subject to metabolic regulation by proteolysis (36Varshavsky A. Genes Cells. 1997; 2: 13-28Crossref PubMed Scopus (267) Google Scholar), employing the cytosolic ubiquitin-proteasome system (36Varshavsky A. Genes Cells. 1997; 2: 13-28Crossref PubMed Scopus (267) Google Scholar). Diabetes is a pathophysiologic condition associated with increased protein degradation, fatty acid oxidation, CPTI activity, and decreased malonyl-CoA inhibition of CPTI (33Park E.A. Mynatt R.L. Cook G.A. Kashfi K. Biochem. J. 1995; 310: 853-858Crossref PubMed Scopus (80) Google Scholar). Thus limited in vivo proteolysis of L-CPTI induced by diabetes, such as cleavage of the first 6 N-terminal residues, may decrease malonyl-CoA sensitivity and alter the normal control of hepatic fatty acid oxidation. Insulin inhibits proteasome activity resulting in decreased cellular protein degradation and controlled fatty acid oxidation (8Hamel F.G. Bennett R.G. Harmon K.S. Duckworth W.C. Biochem. Biophys. Res. Commun. 1997; 234: 671-674Crossref PubMed Scopus (35) Google Scholar). We are currently conducting in vitro partial proteolysis studies with yeast-expressed L-CPTI to determine the role of protein degradation on malonyl-CoA sensitivity. Carnitine palmitoyltransferase I (CPTI)1 catalyzes the conversion of long chain acyl-CoA to acylcarnitines in the presence ofl-carnitine, the first reaction in the transport of long chain fatty acids from the cytoplasm to the mitochondria, a rate-limiting step in β-oxidation (1Bieber L.L. Annu. Rev. Biochem. 1988; 57: 261-283Crossref PubMed Scopus (667) Google Scholar, 2McGarry J.D. Woeltje K.F. Kuwajima M. Foster D.W. Diabetes Metab. Rev. 1989; 5: 271-284Crossref PubMed Scopus (287) Google Scholar). Mammalian tissues express two isoforms of CPTI, a liver isoform (L-CPTI) and a heart/skeletal muscle isoform (M-CPTI), that are 62% identical in amino acid sequence (GenBankTM accession number U62317; Refs. 3Weis B.C. Esser V. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 18712-18715Abstract Full Text PDF PubMed Google Scholar, 4Weis B.C. Cowan A.T. Brown N. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 26443-26448Abstract Full Text PDF PubMed Google Scholar, 5Brown N.F. Weis B.C. Husti J.E. Foster D.W. McGarry J.D. J. Biol. Chem. 1995; 270: 8952-8957Crossref PubMed Scopus (136) Google Scholar, 6Zhu H. Shi J. de Vries Y. Arvidson D.N. Cregg J.M. Woldegiorgis G. Arch. Biochem. Biophys. 1997; 347: 53-61Crossref PubMed Scopus (51) Google Scholar, 7Yamazaki N. Shinhara Y. Shima A. Terada H. FEBS Lett. 1995; 363: 41-45Crossref PubMed Scopus (110) Google Scholar and 9Yamazaki N. Shinhara Y. Shima A. Yamanaka Y. Terada H. Biochim. Biophys. Acta. 1996; 1307: 157-161Crossref PubMed Scopus (102) Google Scholar). As an enzyme that catalyzes the first rate-limiting step in fatty acid oxidation, CPTI is regulated by its physiological inhibitor malonyl-CoA (1Bieber L.L. Annu. Rev. Biochem. 1988; 57: 261-283Crossref PubMed Scopus (667) Google Scholar, 2McGarry J.D. Woeltje K.F. Kuwajima M. Foster D.W. Diabetes Metab. Rev. 1989; 5: 271-284Crossref PubMed Scopus (287) Google Scholar), the first intermediate in fatty acid synthesis, suggesting coordinated control of fatty acid oxidation and synthesis. Understanding the molecular mechanism of the regulation of CPTI by malonyl-CoA is important in the design of drugs for control of excessive fatty acid oxidation in diabetes mellitus (10Prentki M. Corkey B.E. Diabetes. 1996; 45: 273-283Crossref PubMed Scopus (0) Google Scholar) and in myocardial ischemia where accumulation of acylcarnitines has been associated with arrhythmias (11Corr P.B. Yamada K.A. Herz. 1995; 20: 156-168PubMed Google Scholar). We developed a novel high level expression system for rat L-CPTI and human heart M-CPTI in the yeast Pichia pastoris, an organism devoid of endogenous CPT activity (6Zhu H. Shi J. de Vries Y. Arvidson D.N. Cregg J.M. Woldegiorgis G. Arch. Biochem. Biophys. 1997; 347: 53-61Crossref PubMed Scopus (51) Google Scholar, 12de Vries Y. Arvidson D.N. Waterham H.R. Cregg J.M. Woldegiorgis G. Biochemistry. 1997; 36: 5285-5292Crossref PubMed Scopus (52) Google Scholar, 13Zhu H. Shi J. Cregg J.M. Woldegiorgis G. Biochem. Biophys. Res. Commun. 1997; 239: 498-502Crossref PubMed Scopus (29) Google Scholar). Using this system, we demonstrated conclusively that L-CPTI and M-CPTI are active, distinct, malonyl-CoA-sensitive CPTIs that are reversibly inactivated by detergents. We recently showed that deletion of the conserved first 18 N-terminal amino acid residues of rat L-CPTI abolishes malonyl-CoA inhibition and high affinity malonyl-CoA binding (14Shi J. Zhu H. Arvidson D.N. Cregg J.M. Woldegiorgis G. Biochemistry. 1998; 37: 11033-11038Crossref PubMed Scopus (44) Google Scholar). In this study, we have constructed and characterized rat L-CPTI deletion mutants of the first 12 and 6 N-terminal amino acid residues. To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we also constructed three substitution mutations within the conserved first 6 N-terminal amino acid residues (Glu3 → Ala, His5 → Ala, and Gln6 → Ala). DISCUSSIONTo determine the role of the first 130 N-terminal amino acid residues of rat L-CPTI in malonyl-CoA sensitivity and binding, we previously constructed a series of deletion mutants and demonstrated that a mutant lacking the first conserved 18 N-terminal amino acid residues had activity and kinetic properties similar to those of wild type L-CPTI but had completely lost malonyl-CoA sensitivity and high affinity binding (14Shi J. Zhu H. Arvidson D.N. Cregg J.M. Woldegiorgis G. Biochemistry. 1998; 37: 11033-11038Crossref PubMed Scopus (44) Google Scholar). Based on these previous studies (14Shi J. Zhu H. Arvidson D.N. Cregg J.M. Woldegiorgis G. Biochemistry. 1998; 37: 11033-11038Crossref PubMed Scopus (44) Google Scholar), we report here on deletion mutations of the conserved first 12 and 6 N-terminal residues of L-CPTI. Like Δ18, Δ12 and Δ6 had 60–70% of the wild type activity and showed loss of both malonyl-CoA sensitivity and high affinity malonyl-CoA binding, indicating that residue(s) essential for malonyl-CoA binding and sensitivity reside within the conserved first 6 N-terminal amino acids. Of these conserved first 6 N-terminal amino acids, including the start codon Met, residues 2 and 4 are Ala, residue 3 is Glu, residue 5 is His, and residue 6 is Gln. Therefore, we constructed mutants with substitutions of Glu3 with Ala, His5 with Ala, and Gln6 with Ala of L-CPTI.The mutant L-CPTI with a replacement of Glu3 with Ala had a phenotype similar to that of the N-terminal deletion mutants. The mutation resulted in complete loss of malonyl-CoA sensitivity and high affinity malonyl-CoA binding and a decrease in the low affinity malonyl-CoA binding. In contrast, substitution of Glu3 with Ala did not have a significant effect on the kinetic properties of the enzyme, because there was no change in the K m value for palmitoyl-CoA and only a slight increase in theK m value for carnitine. The 29–40% loss in catalytic activity observed with the deletion and point mutants compared with the wild type could be due to a reduction in the expression level or lack of interaction of the N-terminal domain with the catalytic domain as a result of the N-terminal mutations. A protein of the expected size (88 kDa) was detected in the mitochondria of the Glu3 → Ala mutant strain on immunoblotting with L-CPTI specific antibodies. These results demonstrate clearly that Glu3 in the wild type L-CPTI is essential for malonyl-CoA inhibition and binding but not for catalysis, because the kinetic properties of the mutant enzyme are virtually indistinguishable from those of the wild type. This is the first report to demonstrate the critical role of Glu3 residue of L-CPTI for malonyl-CoA sensitivity and binding.The high affinity site (K D1,B max1) for binding of malonyl-CoA to L-CPTI was completely abolished in the Glu3 → Ala, Δ6, and Δ18 mutants, suggesting that the >100-fold decrease in malonyl-CoA sensitivity observed in these mutants was due to the loss of the high affinity binding entity of the enzyme. Although low affinity malonyl-CoA binding was weakened, there was no change in theB max2 value between wild type L-CPTI and mutants Glu3 → Ala, Δ6, and Δ18, suggesting that the residual malonyl-CoA sensitivity observed in the mutants was due to the low affinity malonyl-CoA-binding entity of the enzyme. The results of this study provide strong evidence implicating Glu3 as one of the residues involved in high affinity malonyl-CoA binding. We hypothesize that the Glu3 → Ala substitution may disrupt a hydrogen bonding network or a salt bridge, perhaps to a residue near the active site of CPTI. As the high affinity site is abolished and binding to the low affinity site is weakened, the two sites may partially overlap. Alternatively, the possible loss of a salt bridge may weaken K D2 indirectly.Replacement of His5 with Ala had a much less drastic effect on the IC50 for malonyl-CoA inhibition of L-CPTI but severely diminished both high and low affinity malonyl-CoA binding. TheB max1 for this mutant showed a slight increase, but B max2 showed a significant decrease compared with the wild type value, suggesting that the 10-fold lower IC50 for malonyl-CoA inhibition observed with this mutant, compared with mutants Glu3 → Ala, Δ6, and Δ18, may be due to a slight increase in abundance of the high affinity binding entity with a lowered (100-fold) affinity for malonyl-CoA. The decrease in low affinity malonyl-CoA binding observed for the His5→ Ala mutant (∼15-fold increase in K D2) may be due, in part, to the decreased abundance of the low affinity binding entity of the enzyme (∼3-fold decrease in B max2). Because mutation of His5 → Ala reduced the malonyl-CoA sensitivity and binding, L-CPTI may be affected by pH. A pH-induced shift in malonyl-CoA sensitivity has been reported for CPTI (25Stephens T.W. Cook G.A. Harris R.A. Biochem. J. 1983; 212: 521-524Crossref PubMed Scopus (32) Google Scholar, 26Mills S.E. Foster D.W. McGarry J.D. Biochem. J. 1984; 219: 601-608Crossref PubMed Scopus (50) Google Scholar).Our data clearly demonstrate that there are two classes of malonyl-CoA-binding sites in L-CPTI, namely, a high affinity and a low affinity binding site, similar to earlier studies in isolated rat liver and heart mitochondria (26Mills S.E. Foster D.W. McGarry J.D. Biochem. J. 1984; 219: 601-608Crossref PubMed Scopus (50) Google Scholar, 27Bird M.I. Saggerson E.D. Biochem. J. 1984; 222: 639-647Crossref PubMed Scopus (42) Google Scholar). A previous attempt to express a mutant L-CPTI that lacked the first 82 N-terminal residues was described by Brown et al. (28Brown N.F. Esser V. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 26438-26442Abstract Full Text PDF PubMed Google Scholar), but results were inconclusive due to extremely low expression levels (12de Vries Y. Arvidson D.N. Waterham H.R. Cregg J.M. Woldegiorgis G. Biochemistry. 1997; 36: 5285-5292Crossref PubMed Scopus (52) Google Scholar). The residual malonyl-CoA sensitivity shown by the deletion mutants is similar to that observed with yeast-expressed CPTII (12de Vries Y. Arvidson D.N. Waterham H.R. Cregg J.M. Woldegiorgis G. Biochemistry. 1997; 36: 5285-5292Crossref PubMed Scopus (52) Google Scholar), suggesting that for these mutants malonyl-CoA may inhibit via direct interaction with the active site. Additional studies are needed to determine whether the active site acts as a low affinity malonyl-CoA-binding site, but our data suggest that there may be some overlap between the malonyl-CoA and palmitoyl-CoA binding sites. In the absence of malonyl-CoA, free CoA (50 μm) and acetyl-CoA (500 μm) inhibited the activities of both the wild type and the Glu3 → Ala mutant L-CPTI by 50%. 2J. Shi, H. Zhu, D. N. Arvidson, and G. Woldegiorgis, unpublished observation.Because a total loss of the high affinity malonyl-CoA binding site was observed in the Glu3 → Ala mutant, the results suggest that CoA and acetyl-CoA inhibit by binding to the active site or the low affinity malonyl-CoA-binding site. At high concentrations, both CoA and the substrate palmitoyl-CoA reduce the inhibition of L-CPTI by malonyl-CoA (18Bremer J. Woldegiorgis G. Schalinske K. Shrago E. Biochim. Biophys. Acta. 1985; 833: 9-16Crossref PubMed Scopus (67) Google Scholar, 29Cook G.A. Mynatt R.L. Kashfi K. J. Biol. Chem. 1994; 269: 8803-8807Abstract Full Text PDF PubMed Google Scholar), suggesting partial overlap between the malonyl-CoA and the substrate binding sites.Based on limited proteolysis studies of intact and outer membrane rat liver mitochondria, a model for the membrane topology of L-CPTI has been proposed that predicts exposure of 90% of L-CPTI, including N and C termini domains crucial for activity and malonyl-CoA sensitivity of the enzyme on the cytosolic side of the outer mitochondrial membrane (30Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (122) Google Scholar). A more recent detailed deletion mutation analysis study of the 129 N-terminal amino acid residues of the yeast-expressed L-CPTI from our laboratory clearly demonstrated that residues critical for malonyl-CoA inhibition and binding of L-CPTI are located within the conserved first 18 N-terminal amino acid residues of the enzyme (14Shi J. Zhu H. Arvidson D.N. Cregg J.M. Woldegiorgis G. Biochemistry. 1998; 37: 11033-11038Crossref PubMed Scopus (44) Google Scholar). In this study, we demonstrate that glutamic acid residue 3 and histidine 5 are essential for malonyl-CoA binding and inhibition.Limited proteolysis of intact and outer membrane preparations of rat liver mitochondria result in a marked loss in L-CPTI activity and malonyl-CoA sensitivity (30Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (122) Google Scholar, 31Kashfi K. Mynatt R.L. Cook G.A. Biochim. Biophys. Acta. 1994; 1212: 245-252Crossref PubMed Scopus (31) Google Scholar), accompanied by the cleavage of the extreme N terminus (<1 kDa) of L-CPTI (30Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (122) Google Scholar). Mitochondria isolated from fasted and diabetic rat livers, metabolic conditions with increased fatty acid oxidation, exhibit increased L-CPTI activity and decreased malonyl-CoA sensitivity (32Cook G.A. Gamble M.S. J. Biol. Chem. 1987; 262: 2050-2055Abstract Full Text PDF PubMed Google Scholar). Furthermore, insulin reverses the effects of diabetes on L-CPTI activity and malonyl-CoA sensitivity (32Cook G.A. Gamble M.S. J. Biol. Chem. 1987; 262: 2050-2055Abstract Full Text PDF PubMed Google Scholar). Thus, fasting and diabetes, metabolic conditions that enhance protein degradation, reduce the sensitivity of CPTI to malonyl-CoA inhibition (32Cook G.A. Gamble M.S. J. Biol. Chem. 1987; 262: 2050-2055Abstract Full Text PDF PubMed Google Scholar, 33Park E.A. Mynatt R.L. Cook G.A. Kashfi K. Biochem. J. 1995; 310: 853-858Crossref PubMed Scopus (80) Google Scholar, 34Kashfi K. Cagen L. Cook G.A. Lipids. 1995; 30: 383-388Crossref PubMed Scopus (5) Google Scholar). The L-CPTI gene in INS-1 cells may be an early response gene like c-fos (35Assimacopoulos-Jeannet F. Thumelin S. Roche E. Esser V. McGarry J.D. Prentki M. J. Biol. Chem. 1997; 272: 1659-1664Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), suggesting that the enzyme may be subject to metabolic regulation by proteolysis (36Varshavsky A. Genes Cells. 1997; 2: 13-28Crossref PubMed Scopus (267) Google Scholar), employing the cytosolic ubiquitin-proteasome system (36Varshavsky A. Genes Cells. 1997; 2: 13-28Crossref PubMed Scopus (267) Google Scholar). Diabetes is a pathophysiologic condition associated with increased protein degradation, fatty acid oxidation, CPTI activity, and decreased malonyl-CoA inhibition of CPTI (33Park E.A. Mynatt R.L. Cook G.A. Kashfi K. Biochem. J. 1995; 310: 853-858Crossref PubMed Scopus (80) Google Scholar). Thus limited in vivo proteolysis of L-CPTI induced by diabetes, such as cleavage of the first 6 N-terminal residues, may decrease malonyl-CoA sensitivity and alter the normal control of hepatic fatty acid oxidation. Insulin inhibits proteasome activity resulting in decreased cellular protein degradation and controlled fatty acid oxidation (8Hamel F.G. Bennett R.G. Harmon K.S. Duckworth W.C. Biochem. Biophys. Res. Commun. 1997; 234: 671-674Crossref PubMed Scopus (35) Google Scholar). We are currently conducting in vitro partial proteolysis studies with yeast-expressed L-CPTI to determine the role of protein degradation on malonyl-CoA sensitivity. To determine the role of the first 130 N-terminal amino acid residues of rat L-CPTI in malonyl-CoA sensitivity and binding, we previously constructed a series of deletion mutants and demonstrated that a mutant lacking the first conserved 18 N-terminal amino acid residues had activity and kinetic properties similar to those of wild type L-CPTI but had completely lost malonyl-CoA sensitivity and high affinity binding (14Shi J. Zhu H. Arvidson D.N. Cregg J.M. Woldegiorgis G. Biochemistry. 1998; 37: 11033-11038Crossref PubMed Scopus (44) Google Scholar). Based on these previous studies (14Shi J. Zhu H. Arvidson D.N. Cregg J.M. Woldegiorgis G. Biochemistry. 1998; 37: 11033-11038Crossref PubMed Scopus (44) Google Scholar), we report here on deletion mutations of the conserved first 12 and 6 N-terminal residues of L-CPTI. Like Δ18, Δ12 and Δ6 had 60–70% of the wild type activity and showed loss of both malonyl-CoA sensitivity and high affinity malonyl-CoA binding, indicating that residue(s) essential for malonyl-CoA binding and sensitivity reside within the conserved first 6 N-terminal amino acids. Of these conserved first 6 N-terminal amino acids, including the start codon Met, residues 2 and 4 are Ala, residue 3 is Glu, residue 5 is His, and residue 6 is Gln. Therefore, we constructed mutants with substitutions of Glu3 with Ala, His5 with Ala, and Gln6 with Ala of L-CPTI. The mutant L-CPTI with a replacement of Glu3 with Ala had a phenotype similar to that of the N-terminal deletion mutants. The mutation resulted in complete loss of malonyl-CoA sensitivity and high affinity malonyl-CoA binding and a decrease in the low affinity malonyl-CoA binding. In contrast, substitution of Glu3 with Ala did not have a significant effect on the kinetic properties of the enzyme, because there was no change in the K m value for palmitoyl-CoA and only a slight increase in theK m value for carnitine. The 29–40% loss in catalytic activity observed with the deletion and point mutants compared with the wild type could be due to a reduction in the expression level or lack of interaction of the N-terminal domain with the catalytic domain as a result of the N-terminal mutations. A protein of the expected size (88 kDa) was detected in the mitochondria of the Glu3 → Ala mutant strain on immunoblotting with L-CPTI specific antibodies. These results demonstrate clearly that Glu3 in the wild type L-CPTI is essential for malonyl-CoA inhibition and binding but not for catalysis, because the kinetic properties of the mutant enzyme are virtually indistinguishable from those of the wild type. This is the first report to demonstrate the critical role of Glu3 residue of L-CPTI for malonyl-CoA sensitivity and binding. The high affinity site (K D1,B max1) for binding of malonyl-CoA to L-CPTI was completely abolished in the Glu3 → Ala, Δ6, and Δ18 mutants, suggesting that the >100-fold decrease in malonyl-CoA sensitivity observed in these mutants was due to the loss of the high affinity binding entity of the enzyme. Although low affinity malonyl-CoA binding was weakened, there was no change in theB max2 value between wild type L-CPTI and mutants Glu3 → Ala, Δ6, and Δ18, suggesting that the residual malonyl-CoA sensitivity observed in the mutants was due to the low affinity malonyl-CoA-binding entity of the enzyme. The results of this study provide strong evidence implicating Glu3 as one of the residues involved in high affinity malonyl-CoA binding. We hypothesize that the Glu3 → Ala substitution may disrupt a hydrogen bonding network or a salt bridge, perhaps to a residue near the active site of CPTI. As the high affinity site is abolished and binding to the low affinity site is weakened, the two sites may partially overlap. Alternatively, the possible loss of a salt bridge may weaken K D2 indirectly. Replacement of His5 with Ala had a much less drastic effect on the IC50 for malonyl-CoA inhibition of L-CPTI but severely diminished both high and low affinity malonyl-CoA binding. TheB max1 for this mutant showed a slight increase, but B max2 showed a significant decrease compared with the wild type value, suggesting that the 10-fold lower IC50 for malonyl-CoA inhibition observed with this mutant, compared with mutants Glu3 → Ala, Δ6, and Δ18, may be due to a slight increase in abundance of the high affinity binding entity with a lowered (100-fold) affinity for malonyl-CoA. The decrease in low affinity malonyl-CoA binding observed for the His5→ Ala mutant (∼15-fold increase in K D2) may be due, in part, to the decreased abundance of the low affinity binding entity of the enzyme (∼3-fold decrease in B max2). Because mutation of His5 → Ala reduced the malonyl-CoA sensitivity and binding, L-CPTI may be affected by pH. A pH-induced shift in malonyl-CoA sensitivity has been reported for CPTI (25Stephens T.W. Cook G.A. Harris R.A. Biochem. J. 1983; 212: 521-524Crossref PubMed Scopus (32) Google Scholar, 26Mills S.E. Foster D.W. McGarry J.D. Biochem. J. 1984; 219: 601-608Crossref PubMed Scopus (50) Google Scholar). Our data clearly demonstrate that there are two classes of malonyl-CoA-binding sites in L-CPTI, namely, a high affinity and a low affinity binding site, similar to earlier studies in isolated rat liver and heart mitochondria (26Mills S.E. Foster D.W. McGarry J.D. Biochem. J. 1984; 219: 601-608Crossref PubMed Scopus (50) Google Scholar, 27Bird M.I. Saggerson E.D. Biochem. J. 1984; 222: 639-647Crossref PubMed Scopus (42) Google Scholar). A previous attempt to express a mutant L-CPTI that lacked the first 82 N-terminal residues was described by Brown et al. (28Brown N.F. Esser V. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 26438-26442Abstract Full Text PDF PubMed Google Scholar), but results were inconclusive due to extremely low expression levels (12de Vries Y. Arvidson D.N. Waterham H.R. Cregg J.M. Woldegiorgis G. Biochemistry. 1997; 36: 5285-5292Crossref PubMed Scopus (52) Google Scholar). The residual malonyl-CoA sensitivity shown by the deletion mutants is similar to that observed with yeast-expressed CPTII (12de Vries Y. Arvidson D.N. Waterham H.R. Cregg J.M. Woldegiorgis G. Biochemistry. 1997; 36: 5285-5292Crossref PubMed Scopus (52) Google Scholar), suggesting that for these mutants malonyl-CoA may inhibit via direct interaction with the active site. Additional studies are needed to determine whether the active site acts as a low affinity malonyl-CoA-binding site, but our data suggest that there may be some overlap between the malonyl-CoA and palmitoyl-CoA binding sites. In the absence of malonyl-CoA, free CoA (50 μm) and acetyl-CoA (500 μm) inhibited the activities of both the wild type and the Glu3 → Ala mutant L-CPTI by 50%. 2J. Shi, H. Zhu, D. N. Arvidson, and G. Woldegiorgis, unpublished observation.Because a total loss of the high affinity malonyl-CoA binding site was observed in the Glu3 → Ala mutant, the results suggest that CoA and acetyl-CoA inhibit by binding to the active site or the low affinity malonyl-CoA-binding site. At high concentrations, both CoA and the substrate palmitoyl-CoA reduce the inhibition of L-CPTI by malonyl-CoA (18Bremer J. Woldegiorgis G. Schalinske K. Shrago E. Biochim. Biophys. Acta. 1985; 833: 9-16Crossref PubMed Scopus (67) Google Scholar, 29Cook G.A. Mynatt R.L. Kashfi K. J. Biol. Chem. 1994; 269: 8803-8807Abstract Full Text PDF PubMed Google Scholar), suggesting partial overlap between the malonyl-CoA and the substrate binding sites. Based on limited proteolysis studies of intact and outer membrane rat liver mitochondria, a model for the membrane topology of L-CPTI has been proposed that predicts exposure of 90% of L-CPTI, including N and C termini domains crucial for activity and malonyl-CoA sensitivity of the enzyme on the cytosolic side of the outer mitochondrial membrane (30Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (122) Google Scholar). A more recent detailed deletion mutation analysis study of the 129 N-terminal amino acid residues of the yeast-expressed L-CPTI from our laboratory clearly demonstrated that residues critical for malonyl-CoA inhibition and binding of L-CPTI are located within the conserved first 18 N-terminal amino acid residues of the enzyme (14Shi J. Zhu H. Arvidson D.N. Cregg J.M. Woldegiorgis G. Biochemistry. 1998; 37: 11033-11038Crossref PubMed Scopus (44) Google Scholar). In this study, we demonstrate that glutamic acid residue 3 and histidine 5 are essential for malonyl-CoA binding and inhibition. Limited proteolysis of intact and outer membrane preparations of rat liver mitochondria result in a marked loss in L-CPTI activity and malonyl-CoA sensitivity (30Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (122) Google Scholar, 31Kashfi K. Mynatt R.L. Cook G.A. Biochim. Biophys. Acta. 1994; 1212: 245-252Crossref PubMed Scopus (31) Google Scholar), accompanied by the cleavage of the extreme N terminus (<1 kDa) of L-CPTI (30Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (122) Google Scholar). Mitochondria isolated from fasted and diabetic rat livers, metabolic conditions with increased fatty acid oxidation, exhibit increased L-CPTI activity and decreased malonyl-CoA sensitivity (32Cook G.A. Gamble M.S. J. Biol. Chem. 1987; 262: 2050-2055Abstract Full Text PDF PubMed Google Scholar). Furthermore, insulin reverses the effects of diabetes on L-CPTI activity and malonyl-CoA sensitivity (32Cook G.A. Gamble M.S. J. Biol. Chem. 1987; 262: 2050-2055Abstract Full Text PDF PubMed Google Scholar). Thus, fasting and diabetes, metabolic conditions that enhance protein degradation, reduce the sensitivity of CPTI to malonyl-CoA inhibition (32Cook G.A. Gamble M.S. J. Biol. Chem. 1987; 262: 2050-2055Abstract Full Text PDF PubMed Google Scholar, 33Park E.A. Mynatt R.L. Cook G.A. Kashfi K. Biochem. J. 1995; 310: 853-858Crossref PubMed Scopus (80) Google Scholar, 34Kashfi K. Cagen L. Cook G.A. Lipids. 1995; 30: 383-388Crossref PubMed Scopus (5) Google Scholar). The L-CPTI gene in INS-1 cells may be an early response gene like c-fos (35Assimacopoulos-Jeannet F. Thumelin S. Roche E. Esser V. McGarry J.D. Prentki M. J. Biol. Chem. 1997; 272: 1659-1664Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), suggesting that the enzyme may be subject to metabolic regulation by proteolysis (36Varshavsky A. Genes Cells. 1997; 2: 13-28Crossref PubMed Scopus (267) Google Scholar), employing the cytosolic ubiquitin-proteasome system (36Varshavsky A. Genes Cells. 1997; 2: 13-28Crossref PubMed Scopus (267) Google Scholar). Diabetes is a pathophysiologic condition associated with increased protein degradation, fatty acid oxidation, CPTI activity, and decreased malonyl-CoA inhibition of CPTI (33Park E.A. Mynatt R.L. Cook G.A. Kashfi K. Biochem. J. 1995; 310: 853-858Crossref PubMed Scopus (80) Google Scholar). Thus limited in vivo proteolysis of L-CPTI induced by diabetes, such as cleavage of the first 6 N-terminal residues, may decrease malonyl-CoA sensitivity and alter the normal control of hepatic fatty acid oxidation. Insulin inhibits proteasome activity resulting in decreased cellular protein degradation and controlled fatty acid oxidation (8Hamel F.G. Bennett R.G. Harmon K.S. Duckworth W.C. Biochem. Biophys. Res. Commun. 1997; 234: 671-674Crossref PubMed Scopus (35) Google Scholar). We are currently conducting in vitro partial proteolysis studies with yeast-expressed L-CPTI to determine the role of protein degradation on malonyl-CoA sensitivity. We are grateful to Dr. James M. Cregg (Oregon Graduate Institute of Science and Technology) for advice, helpful suggestions, and encouragement throughout these studies." @default.
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W1963817811 title "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" @default.
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