Mutagenesis of specific amino acids converts carnitine acetyltransferase into carnitine palmitoyltransferase

Carnitine acyltransferases catalyze the exchange of acyl groups between carnitine and CoA. The members of the family can be classified on the basis of their acyl-CoA selectivity. Carnitine acetyltransferases (CrATs) are very active toward short-chain acyl-CoAs but not toward medium- or long-chain acyl-CoAs. Previously, we identified an amino acid residue (Met(564) in rat CrAT) that was critical to fatty acyl-chain-length specificity. M564G-mutated CrAT behaved as if its natural substrates were medium-chain acyl-CoAs, similar to that of carnitine octanoyltransferase (COT). To extend the specificity of rat CrAT to other substrates, we have performed new mutations. Using in silico molecular modeling procedures, we have now identified a second putative amino acid involved in acyl-CoA specificity (Asp(356) in rat CrAT). The double CrAT mutant D356A/M564G showed 6-fold higher activity toward palmitoyl-CoA than that of the single CrAT mutant M564G and a new activity toward stearoyl-CoA. We show that by performing two amino acid replacements a CrAT can be converted into a pseudo carnitine palmitoyltransferase (CPT) in terms of substrate specificity. To change CrAT specificity from carnitine to choline, we also prepared a mutant CrAT that incorporates four amino acid substitutions (A106M/T465V/T467N/R518N). The quadruple mutant shifted the catalytic discrimination between l-carnitine and choline in favor of the latter substrate and showed a 9-fold increase in catalytic efficiency toward choline compared with that of the wild-type. Molecular in silico docking supports kinetic data for the positioning of substrates in the catalytic site of CrAT mutants.     

Structural and biochemical studies of the substrate selectivity of carnitine acetyltransferase

Carnitine acyltransferases catalyze the exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids, and are attractive targets for drug discovery against diabetes and obesity. These enzymes are classified based on their substrate selectivity for short-chain, medium-chain, or long-chain fatty acids. Structural information on carnitine acetyltransferase suggests that residues Met-564 and Phe-565 may be important determinants of substrate selectivity with the side chain of Met-564 located in the putative binding pocket for acyl groups. Both residues are replaced by glycine in carnitine palmitoyltransferases. To assess the functional relevance of this structural observation, we have replaced these two residues with small amino acids by mutagenesis, characterized the substrate preference of the mutants, and determined the crystal structures of two of these mutants. Kinetic studies confirm that the M564G or M564A mutation is sufficient to increase the activity of the enzyme toward medium-chain substrates with hexanoyl-CoA being the preferred substrate for the M564G mutant. The crystal structures of the M564G mutant, both alone and in complex with carnitine, reveal a deep binding pocket that can accommodate the larger acyl group. We have determined the crystal structure of the F565A mutant in a ternary complex with both the carnitine and CoA substrates at a 1.8-A resolution. The F565A mutation has minor effects on the structure or the substrate preference of the enzyme.     

Redesign of carnitine acetyltransferase specificity by protein engineering

In eukaryotes, L-carnitine is involved in energy metabolism by facilitating beta-oxidation of fatty acids. Carnitine acetyltransferases (CrAT) catalyze the reversible conversion of acetyl-CoA and carnitine to acetylcarnitine and free CoA. To redesign the specificity of rat CrAT toward its substrates, we mutated Met564. The M564G mutated CrAT showed higher activity toward longer chain acyl-CoAs: activity toward myristoyl-CoA was 1250-fold higher than that of the wild-type CrAT, and lower activity toward its natural substrate, acetyl-CoA. Kinetic constants of the mutant CrAT showed modification in favor of longer acyl-CoAs as substrates. In the reverse case, mutation of the orthologous glycine (Gly553) to methionine in carnitine octanoyltransferase (COT) decreased activity toward its natural substrates, medium- and long-chain acyl-CoAs, and increased activity toward short-chain acyl-CoAs. Another CrAT mutant, M564A, was prepared and tested in the same way, with similar results. We conclude that Met564 blocks the entry of medium- and long-chain acyl-CoAs to the catalytic site of CrAT. Three-dimensional models of wild-type and mutated CrAT and COT support this hypothesis. We show for the first time that a single amino acid is able to determine the substrate specificity of CrAT and COT.     

Crystal structure of carnitine acetyltransferase and implications for the catalytic mechanism and fatty acid transport

Carnitine acyltransferases have crucial roles in the transport of fatty acids for beta-oxidation. Dysregulation of these enzymes can lead to serious diseases in humans, and they are targets for therapeutic development against diabetes. We report the crystal structures of murine carnitine acetyltransferase (CRAT), alone and in complex with its substrate carnitine or CoA. The structure contains two domains. Surprisingly, these two domains share the same backbone fold, which is also similar to that of chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. The active site is located at the interface between the two domains. Carnitine and CoA are bound in deep channels in the enzyme, on opposite sides of the catalytic His343 residue. The structural information provides a molecular basis for understanding the catalysis by carnitine acyltransferases and for designing their inhibitors. Specifically, our structural information suggests that the substrate carnitine may assist the catalysis by stabilizing the oxyanion in the reaction intermediate.     

Inhibition of carnitine acetyltransferase by mildronate,a regulator of energy metabolism

Carnitine acetyltransferase (CrAT; EC 2.3.1.7) catalyzes the reversible transfer of acetyl groups between acetyl-coenzyme A (acetyl-CoA) and L-carnitine; it also regulates the cellular pool of CoA and the availability of activated acetyl groups. In this study, biochemical measurements, saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, and molecular docking were applied to give insights into the CrAT binding of a synthetic inhibitor, the cardioprotective drug mildronate (3-(2,2,2-trimethylhydrazinium)-propionate). The obtained results show that mildronate inhibits CrAT in a competitive manner through binding to the carnitine binding site, not the acetyl-CoA binding site. The bound conformation of mildronate closely resembles that of carnitine except for the orientation of the trimethylammonium group, which in the mildronate molecule is exposed to the solvent. The dissociation constant of the mildronate CrAT complex is approximately 0.1?mM, and the K_i is 1.6?mM. The results suggest that the cardioprotective effect of mildronate might be partially mediated by CrAT inhibition and concomitant regulation of cellular energy metabolism pathways.     

Substrate specificity of human carnitine acetyltransferase: Implications for fatty acid and branched-chain amino acid metabolism

Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.     

Structure of human carnitine acetyltransferase. Molecular basis for fatty acyl transfer

Carnitine acyltransferases are a family of ubiquitous enzymes that play a pivotal role in cellular energy metabolism. We report here the x-ray structure of human carnitine acetyltransferase to a 1.6-A resolution. This structure reveals a monomeric protein of two equally sized alpha/beta domains. Each domain is shown to have a partially similar fold to other known but oligomeric enzymes that are also involved in group-transfer reactions. The unique monomeric arrangement of the two domains constitutes a central narrow active site tunnel, indicating a likely universal feature for all members of the carnitine acyltransferase family. Superimposition of the substrate complex of a related protein, dihydrolipoyl trans-acetylase, reveals that both substrates localize to the active site tunnel of human carnitine acetyltransferase, suggesting the location of the ligand binding sites for carnitine and coenzyme A. Most significantly, this structure provides critical insights into the molecular basis for fatty acyl chain transfer and a possible common mechanism among a wide range of acyltransferases utilizing a catalytic dyad.     

Biosynthesis of phosphatidic acid by glycerophosphate acyltransferases in rat liver mitochondria and microsomes

The acyltransferases that catalyze the synthesis of phosphatidic acid from labelled sn-[14C]glycero-3-phosphate and fatty acyl carnitine or coenzyme A derivatives have been shown to be present in both isolated mitochondria and microsomes from rat liver. The major reaction product was phosphatidic acid in both subcellular fractions. A small quantity of lysophosphatidic acid and neutral lipids were produced as by-products. Divalent cations had significant effects on both mitochondrial and microsomal fractions in stimulating acylation using palmitoyl CoA, but not when palmitoyl carnitine was used as the acyl donor. Palmitoyl CoA and palmitoyl carnitine could be used for acylation by both mitochondria and microsomes. Mitochondria were more permeable to palmitoyl carnitine and readily used it as the substrate for acylation. On the other hand, microsomes yielded a better rate with palmitoyl CoA and the rate of acylation from palmitoyl carnitine in microsomes was correlated with the degree of mitochondrial contamination. The enzymes were partially purified from Triton X-100 extracts of subcellular fractions. Based on the differences of substrate utilization, products formed, divalent cation effects, molecular weights, and polarity, the mitochondrial and microsomal acyltransferases appeared to be different enzymes.     

Some kinetic studies on the mechanism of action of carnitine acetyltransferase

1. Michaelis constants for substrates of carnitine acetyltransferase have been shown to be independent of the concentration of second substrate present. This applies to the forward reaction between acetyl-l-carnitine and CoASH, and to the back reaction between l-carnitine and acetyl-CoA. 2. Product inhibition of both forward and back reactions has been studied. Evidence has been obtained for independent binding sites for l-carnitine and CoASH. Acetyl groups attached to either substrate occupy overlapping positions in space when the substrates are bound to the enzyme. 3. Possible reaction mechanisms involving the ordered addition of substrates have been excluded by determining kinetic constants in the presence and absence of added product. 4. d-Carnitine and acetyl-d-carnitine have been shown to inhibit competitively with respect to l-carnitine and acetyl-l-carnitine. 5. It is concluded that the mechanism of action of carnitine acetyltransferase involves four binary and two or more ternary enzyme complexes in rapid equilibrium with free substrates, the interconversion of the ternary complexes being the rate-limiting step. The possible intermediate formation of an acetyl-enzyme cannot be excluded, but this could only arise from a ternary complex.     

Crystal structure of rat carnitine palmitoyltransferase II (CPT-II)

Carnitine palmitoyltransferase II (CPT-II) has a crucial role in the beta-oxidation of long-chain fatty acids in mitochondria. We report here the crystal structure of rat CPT-II at 1.9A resolution. The overall structure shares strong similarity to those of short- and medium-chain carnitine acyltransferases, although detailed structural differences in the active site region have a significant impact on the substrate selectivity of CPT-II. Three aliphatic chains, possibly from a detergent that is used for the crystallization, were found in the structure. Two of them are located in the carnitine and CoA binding sites, respectively. The third aliphatic chain may mimic the long-chain acyl group in the substrate of CPT-II. The binding site for this aliphatic chain does not exist in the short- and medium-chain carnitine acyltransferases, due to conformational differences among the enzymes. A unique insert in CPT-II is positioned on the surface of the enzyme, with a highly hydrophobic surface. It is likely that this surface patch mediates the association of CPT-II with the inner membrane of the mitochondria.     

Random-order ternary complex reaction mechanism of serine acetyltransferase from Escherichia coli

Although serine acetyltransferase (SAT) from Escherichia coli is homologous with a number of bacterial enzymes that catalyze O-acetyl transfer by a sequential (ternary complex) mechanism, it has been suggested, from experiments with the nearly identical enzyme from Salmonella typhimurium, that the reaction could proceed via an acetyl-enzyme intermediate. To resolve the matter, the E. coli gene for SAT was overexpressed and the enzyme purified 13-fold to homogeneity. The results of a steady-state kinetic analysis of the forward reaction are diagnostic for a ternary complex mechanism, and the response of SAT to dead-end inhibitors indicates a random order for the addition of substrates. The linearity of primary double-reciprocal plots, in the presence and absence of dead-end inhibitors, argues that interconversion of ternary complexes is not significantly faster than kcat, whereas substrate inhibition by serine suggests that breakdown of the SAT.CoA binary complex is rate-determining. The results of equilibrium isotope exchange experiments, for both half-reactions, rule out a "ping-pong" mechanism involving an acetyl-enzyme intermediate, and a pre-steady-state kinetic analysis of the turnover of AcCoA supports such a conclusion. Kinetic data for the reverse reaction (acetylation of CoA by O-acetylserine) are also consistent with a steady-state random-order mechanism, wherein both the breakdown of the SAT*serine complex and the interconversion of ternary complexes are partially rate-determining.     

Carnitine acyltransferases and their influence on CoA pools in health and disease

Cells contain limited and sequestered pools of Coenzyme A (CoA) that are essential for activating carboxylate metabolites. Some acyl-CoA esters have high metabolic and signalling impact, so control of CoA ester concentrations is important. This and transfer of the activated acyl moieties between cell compartments without wasting energy on futile cycles of hydrolysis and resynthesis is achieved through the carnitine system. The location, properties of and deficiencies in the carnitine acyltransferases are described in relation to their influence on the CoA pools in the cell and, hence, on metabolism. The protection of free CoA pools in disease states is achieved by excretion of acyl-carnitine so that carnitine supplementation is required where unwanted acyl groups build up, such as in some inherited disorders of fatty acid oxidation. Acetyl-carnitine improves cognition in the brain and propionyl-carnitine improves cardiac performance in heart disease and diabetes. The therapeutic effects of carnitine and its esters are discussed in relation to the integrative influence of the carnitine system across CoA pools. Recent evidence for sequestered pools of activated acetate for synthesis of malonyl-CoA, for the synthesis of polyunsaturated fatty acids and for the inhibition of carnitine palmitoyltransferase 1 to regulate fatty acid oxidation is reviewed.     

Crystal structure of mouse carnitine octanoyltransferase and molecular determinants of substrate selectivity

Carnitine acyltransferases have crucial functions in fatty acid metabolism. Members of this enzyme family show distinctive substrate preferences for short-, medium- or long-chain fatty acids. The molecular mechanism for this substrate selectivity is not clear as so far only the structure of carnitine acetyltransferase has been determined. To further our understanding of these important enzymes, we report here the crystal structures at up to 2.0-A resolution of mouse carnitine octanoyltransferase alone and in complex with the substrate octanoylcarnitine. The structures reveal significant differences in the acyl group binding pocket between carnitine octanoyltransferase and carnitine acetyltransferase. Amino acid substitutions and structural changes produce a larger hydrophobic pocket that binds the octanoyl group in an extended conformation. Mutation of a single residue (Gly-553) in this pocket can change the substrate preference between short- and medium-chain acyl groups. The side chains of Cys-323 and Met-335 at the bottom of this pocket assume dual conformations in the substrate complex, and mutagenesis studies suggest that the Met-335 residue is important for catalysis.     

Crystal structure of Escherichia coli crotonobetainyl-CoA: carnitine CoA-transferase (CaiB) and its complexes with CoA and carnitinyl-CoA

L-Carnitine (R-[-]-3-hydroxy-4-trimethylaminobutyrate) is found in both eukaryotic and prokaryotic cells and participates in diverse processes including long-chain fatty-acid transport and osmoprotection. The enzyme crotonobetainyl/gamma-butyrobetainyl-CoA:carnitine CoA-transferase (CaiB; E.C. 2.8.3.-) catalyzes the first step in carnitine metabolism, leading to the final product gamma-butyrobetaine. The crystal structures of Escherichia coli apo-CaiB, as well as its Asp169Ala mutant bound to CoA and to carnitinyl-CoA, have been determined and refined to 1.6, 2.4, and 2.4 A resolution, respectively. CaiB is composed of two identical circular chains that together form an intertwined dimer. Each monomer consists of a large domain, containing a Rossmann fold, and a small domain. The monomer and dimer resemble those of formyl-CoA transferase from Oxalobacter formigenes, as well as E. coli YfdW, a putative type-III CoA transferase of unknown function. The CoA cofactor-binding site is formed at the interface of the large domain of one monomer and the small domain from the second monomer. Most of the protein-CoA interactions are formed with the Rossmann fold domain. While the location of cofactor binding is similar in the three proteins, the specific CoA-protein interactions vary somewhat between CaiB, formyl-CoA transferase, and YfdW. CoA binding results in a change in the relative positions of the large and small domains compared with apo-CaiB. The observed carnitinyl-CoA product in crystals of the CaiB Asp169Ala mutant cocrystallized with crotonoyl-CoA and carnitine could result from (i) a catalytic mechanism involving a ternary enzyme-substrate complex, independent of a covalent anhydride intermediate with Asp169, (ii) a spontaneous reaction of the substrates in solution, followed by binding to the enzyme, or (iii) an involvement of another residue substituting functionally for Asp169, such as Glu23.     

Structure of the E. coli bifunctional GlmU acetyltransferase active site with substrates and products

The biosynthesis of UDP-GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA-dependent acetylation of GlcN-1-PO(4) to form GlcNAc-1-PO(4) and its subsequent condensation with UTP to form pyrophosphate and UDP-GlcNAc. As a metabolite, UDP-GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl-CoA, CoA/GlcN-1-PO(4), and desulpho-CoA/GlcNAc-1-PO(4). These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2-amino group of GlcN-1-PO(4) is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism.     

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

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

The carnitine acyltransferases: modulators of acyl-CoA-dependent reactions

Carnitine and carnitine acyltransferases were thought to be merely a mechanism for the rapid transfer of activated long-chain fatty acids into the mitochondrion for beta-oxidation, until enzymologists came along. By kinetic, physical and localization studies, eight different mammalian carnitine acyltransferases have been characterized. Of these, five have been cloned and sequenced. The carnitine :acylcarnitine exchange carrier, first characterized in mitochondria, has now been demonstrated immunologically in peroxisomal membranes too. This cell-wide carnitine system consisting of at least six proteins linking at least four intracellular pools of acyl-CoA that supply a multitude of lipid metabolic pathways is clearly more complex than was first thought. In this article, I describe the location and properties of the components to show how they can modulate acyl-CoA-dependent reactions in the cell.     

Comparison of the carnitine acyltransferase activities from rat liver peroxisomes and microsomes

Carnitine acyltransferase activities for acetyl- and octanoyl-CoA (coenzyme A) occur in isolated peroxisomal, mitochondrial, and microsomal fractions from rat and pig liver. Solubility studies indicated that both peroxisomal carnitine acyltransferases were in the soluble matrix. In contrast, the microsomal carnitine acyltransferases were tightly associated with their membrane. The microsomal short-chain transferase, carnitine acetyltransferase, was solubilized and stabilized by extensive treatment of the membrane with 0.4 m KCl or 0.3 m sucrose in 0.1 m pyrophosphate at pH 7.5. The same treatment only partially solubilized the microsomal medium-chain transferase, carnitine octanoyltransferase.Although half of the total carnitine acetyltransferase activity in rat liver resides in peroxisomes and microsomes, previous reports have only investigated the mitochondrial activity. Transferase activity for acetyl- and octanoyl-CoA were about equal in peroxisomal and in microsomal fractions. A 200-fold purification of peroxisomal and microsomal carnitine acetyltransferases was achieved using O-(diethylaminoethyl)-cellulose and cellulose phosphate chromatography. This short-chain transferase preparation contained less than 5% as much carnitine octanoyltransferase and acyl-CoA deacylase activities. This fact, plus differences in solubility and stability of the microsomal transferase system for acetyl- and octanoyl-CoA indicate the existence of two separate enzymes: a carnitine acetyltransferase and a carnitine octanoyltransferase in peroxisomes and in microsomes.Peroxisomal and microsomal carnitine acetyltransferases had similar properties and could be the same protein. They showed identical chromatographic behavior and had the same pH activity profiles and major isoelectric points. They also had the same apparent molecular weight by gel filtration (59,000) and the same relative velocities and K_m values for several short-chain acyl-CoA substrates. Both were active with propionyl-, acetyl-, malonyl-, and acetyacetyl-CoA, but not with succinyl- and β-hydroxy-β-methylglutaryl-CoA as substrates.     

Interactions of phospho- and dephosphosuccinyl coenzyme A synthetase with manganous ion and substrates. Studies of manganese complexes by NMR relaxation rates of water protons.

The interactions of substrates with succinyl-CoA synthetase were investigated by measuring the enhancement of the longitudinal water proton relaxation rate (PRR) due to Mn(II) to the enzyme substrate complexes. The binding of Mn(II) to the enzyme was investigated by EPR. The effects of phosphorylating the enzyme on its interactions with Mn(II) and substrates were also examined. Mn(II) binds weakly to dephosphosuccinyl-CoA synthetase (E) at approximately four sites with a KD value of 0.14 mM, and the PRR enhancement of the complex, epsilonb, at 24.3 MHZ and 25 degree is 18.8. The phosphoenzyme (E-P) binds Mn(II) more strongly at approximately four sites with a KD value of 0.74 mM, and only a small change in epsilonb to 18.1. Mm ADP binds to E at one or two sites with K2 = 0.5 muM, the values of epsilont for the ternary E-Mn-ADP complex is 17.0. Free ADP binds about 126 times more weakly to the enzyme than does Mn-ADP. PRR titrations indicated that the values of epsilont for the ternary E-Mn-ADP and (E-P)-Mn-ADP complexes are about the same. Mn-ATP binds very weakly or not at all to (E-P)-Mn.Formation of the ternary complexes of CoA with E-Mn or (E-P)-Mn could be followed by small but significant increases in the PRR enhancement. No ternary complex with succinate could be detected since the addition of succinate had no effect on the PRR enhancement. However, a large decrease in enhancement, at least 2-fold, was observed upon addition of both succinate and CoA. An increase in the PRR enhancement was produced by the interaction of succinyl-CoA with the E-Mn complex. Upper limits of the dissociation constants for CoA from the quaternary E-Mn-ADP-succinate-CoA complex and for succinyl-CoA from the quaternary E-Mn-ADP-succinyl-CoA complex are 390 and 560 muM, respectively. The epsilon values for the quaternary and quinary complexes are 6.4 and 3.1, respectively. The successive occupation of substrate binding sites of succinyl-CoA synthetase produces alterations in the molecular dynamics or in the conformation of the active site (or both), which are accompanied by progressive decreases in the values of epsilon. Thus, the physical parameter used in these studies relects the previously observed catalytic properties of the enzyme system inasmuch as the catalytic function of succinyl-CoA synthetase is potentiated by substrate binding, and catalytic avtivity in partial reactions is maximized as binding sites are successively occupied.     

Substrate binding and catalytic mechanism of human choline acetyltransferase

Choline acetyltransferase (ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine from choline and acetyl-CoA, and its presence is a defining feature of cholinergic neurons. We report the structure of human ChAT to a resolution of 2.2 A along with structures for binary complexes of ChAT with choline, CoA, and a nonhydrolyzable acetyl-CoA analogue, S-(2-oxopropyl)-CoA. The ChAT-choline complex shows which features of choline are important for binding and explains how modifications of the choline trimethylammonium group can be tolerated by the enzyme. A detailed model of the ternary Michaelis complex fully supports the direct transfer of the acetyl group from acetyl-CoA to choline through a mechanism similar to that seen in the serine hydrolases for the formation of an acyl-enzyme intermediate. Domain movements accompany CoA binding, and a surface loop, which is disordered in the unliganded enzyme, becomes localized and binds directly to the phosphates of CoA, stabilizing the complex. Interactions between this surface loop and CoA may function to lower the KM for CoA and could be important for phosphorylation-dependent regulation of ChAT activity.