A catalytic role for histidine 237 in rat mammary gland thioesterase II

The involvement of a histidyl residue in the catalytic mechanism of thioesterase II, a serine active-site enzyme that catalyzes the chain terminating reaction in de novo fatty acid synthesis, has been inferred from studies with the inhibitor diethyl pyrocarbonate. Its likely location has been predicted by identification of conserved residues in related thioesterases and ultimately confirmed by site-directed mutagenesis. Diethyl pyrocarbonate inactivated the enzyme with a second-order rate constant of 49 M-1 s-1 at pH 6, 10 degrees C. Data analysis indicated that although several residues reacted with the reagent, modification of a single residue was responsible for the inactivation. Removal of a single ethoxycarbonyl moiety by treatment with neutral hydroxylamine completely restored enzyme activity. Prior ethoxycarbonylation of the histidyl residue blocked the ability of the active-site serine to react with phenylmethanesulfonyl fluoride. Comparison of the amino acid sequences of five structurally related proteins indicated that only 1 histidine has been completely conserved. Replacement of this residue in rat thioesterase II (His-237) with arginine and leucine by mutagenesis reduced the catalytic activity by 2-3 orders of magnitude. The activity of the mutant thioesterases, unlike that of the wild-type enzyme, was relatively insensitive to inhibition by diethyl pyrocarbonate and phenylmethylsulfonyl fluoride. These studies provide strong evidence that His-237 is involved directly in catalysis and suggest that its role is to increase the nucleophilic character of the active-site Ser-101 by acting as a proton acceptor thus facilitating acylation of the seryl residue. The mechanism appears to share certain common features with the charge-relay system characteristic of other esterases.     

Amino acid sequence of the serine active-site region of the medium-chain S-acyl fatty acid synthetase thioester hydrolase from rat mammary gland

Medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II), a discrete monomeric enzyme of 29 kDa, regulates the product specificity of the de novo lipogenic systems in certain specialized mammalian and avian tissues, such as mammary and uropygial glands. The amino acid sequence of a 57-residue region containing the active site of the rat mammary gland enzyme has been established by a combination of amino acid and cDNA sequencing. Thioesterase II was radiolabeled with the serine esterase inhibitor [1,3-14C]diisopropyl-fluorophosphate and digested sequentially with cyanogen bromide, Staphylococcus aureus V8 protease and trypsin. A radiolabeled tryptic peptide was isolated and sequenced by automated Edman degradation and the location of the active-site residue established. The amino acid sequence was confirmed by sequencing an overlapping, unlabeled peptide, obtained by V8 digestion of the whole enzyme, and by dideoxynucleotide sequencing of a thioesterase II cDNA clone isolated from a lambda gt11 expression library. The active center contains the motif Gly-Xaa-Ser-Xaa-Gly, characteristic of the serine esterase family of enzymes. A seven-residue region around the essential serine of the rat mammary thioesterase II, Phe-Gly-Met-Ser-Phe-Gly-Ser, is completely homologous with a region of the mallard uropygial thioesterase, recently analyzed by cDNA sequencing, indicating that this is likely to be the active site of the avian enzyme. Overall homology between the mammalian and avian enzymes for the 57-amino-acid residue region is 47% and suggests that the two enzymes may share a common evolutionary origin.     

Site-directed mutagenesis studies on the recombinant thioesterase domain of chicken fatty acid synthase expressed in Escherichia coli

The animal fatty acid synthase is a multifunctional protein with a subunit molecular weight of 260,000. We recently reported the expression and characterization of the acyl carrier protein and thioesterase domains of the chicken liver fatty acid synthase in Escherichia coli. In order to gain insight into the mechanism of action of the thioesterase domain, we have replaced the putative active site serine 101 with alanine and cysteine and the conserved histidine 274 with alanine by site-directed mutagenesis. While both the Ser101----Ala and His274----Ala mutant proteins were inactive, the Ser101----Cys mutant enzyme (thiol-thioesterase) retained considerable activity, but the properties of the enzyme were changed from an active serine esterase to an active cysteine esterase, providing strong evidence for the role of Ser101 as the active site nucleophile. In order to further probe into the role of His274, a double mutant was constructed containing both the Ser101----Cys and the His274----Ala mutations. The double-mutant protein was inactive and exhibited diminished reactivity of the Cys-SH to iodoacetamide as compared to that of the Ser101----Cys-thioesterase, suggesting a role of His274 as a general base in withdrawing the proton from the Cys-SH in the thiol-thioesterase or Ser101 in the wild-type enzyme. Incubation of the recombinant thioesterases with [1-14C] palmitoyl-CoA resulted in the incorporation of [1-14C] palmitoyl into the enzyme only in the double mutant, suggesting that Cys-SH of the double mutant is reactive enough to form the palmitoyl-S-enzyme intermediate. This intermediate is not hydrolyzed because of the lack of His274, which is required for the attack of H2O on the acyl enzyme. These results suggest that the catalytic mechanism of the thioesterases may be similar to that of the serine proteases and lipases, which employ a serine-histidine-aspartic acid catalytic triad as part of their catalytic mechanism.     

Identification of the active-site serine in human pancreatic lipase by chemical modification with tetrahydrolipstatin.

A chemical modification approach was used in this study to identify the active site serine residue of human pancreatic lipase. Purified human pancreatic lipase was covalently modified by incubation with [3H], [14C] tetrahydrolipstatin (THL), a potent inhibitor of pancreatic lipase. The radiolabeled lipase was digested with thermolysin, and the peptides were separated by HPLC. A single THL-peptide-adduct was obtained which was identical to that obtained earlier from porcine pancreatic lipase. This pentapeptide with the sequence VIGHS is covalently bound to a THL molecule via the side chain hydroxyl group of the serine unit corresponding to Ser-152 of the lipase. The selective cleavage of the THL-serine bond by mild acid treatment resulted in the formation of the delta-lactone Ro 40-4441 in high yield and clearly proves that THL is attached via an ester bond and with retention of stereochemistry at all chiral centers to the side chain hydroxyl group of Ser-152 of the lipase. The results obtained for human pancreatic lipase corroborate the inhibition mechanism of THL found on the porcine enzyme, and are in full agreement with the identification of the Ser-152 ... His-263 ... Asp-176 catalytic triad in the X-ray structure of human pancreatic lipase.     

Complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase from rat mammary gland

The complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase (thioesterase II) from rat mammary gland is presented. Most of the sequence was derived by analysis of peptide fragments produced by cleavage at methionyl, glutamyl, lysyl, arginyl, and tryptophanyl residues. A small section of the sequence was deduced from a previously analyzed cDNA clone. The protein consists of 260 residues and has a blocked amino-terminal methionine and calculated Mr of 29,212. The carboxy-terminal sequence, verified by Edman degradation of the carboxy-terminal cyanogen bromide fragment and carboxypeptidase Y digestion of the intact thioesterase II, terminates with a serine residue and lacks three additional residues predicted by the cDNA sequence. The native enzyme contains three cysteine residues but no disulfide bridges. The active site serine residue is located at position 101. The rat mammary gland thioesterase II exhibits approximately 40% homology with a thioesterase from mallard uropygial gland, the sequence of which was recently determined by cDNA analysis [Poulose, A.J., Rogers, L., Cheesbrough, T. M., & Kolattukudy, P. E. (1985) J. Biol. Chem. 260, 15953-15958]. Thus the two enzymes may share similar structural features and a common evolutionary origin. The location of the active site in these thioesterases differs from that of other serine active site esterases; indeed, the enzymes do not exhibit any significant homology with other serine esterases, suggesting that they may constitute a separate new family of serine active site enzymes.     

Identification of two serine residues involved in catalysis by fatty acid amide hydrolase.

Fatty acid amide hydrolase is an integral membrane protein that hydrolyzes a novel and growing class of neuromodulatory fatty acid molecules, including anandamide, 2-arachidonyl glycerol, and oleamide. This activity is inhibited by serine and cysteine reactive agents, suggesting that the active site contains a serine or cysteine residue. Therefore serine and cysteine residues were mutated to alanine and the effects on activity were determined. Mutants were prepared using site-directed mutagenesis methods and expressed in COS-7 cells. Serine mutations S217A and S241A completely abolished enzymatic activity. Mutants S152A and C249A had no effect on activity, while S218A showed a slight decrease in activity. To confirm these results biochemically, the mutant enzymes were reacted with the irreversible inhibitor [(14)C]-diisopropyl fluorophosphate. All of the mutants except S217A and S241A were labeled. We therefore confirm that fatty acid amide hydrolase is a serine hydrolase and propose that both Ser-217 and Ser-241 are essential for enzyme activity.     

Highly selective hydrolysis of fatty acyl-CoAs by calcium-independent phospholipase A2beta. Enzyme autoacylation and acyl-CoA-mediated reversal of calmodulin inhibition of phospholipase A2 activity

Calcium-independent phospholipase A2beta (iPLA2beta) participates in numerous diverse cellular processes, such as arachidonic acid release, insulin secretion, calcium signaling, and apoptosis. Herein, we demonstrate the highly selective iPLA2beta-catalyzed hydrolysis of saturated long-chain fatty acyl-CoAs (palmitoyl-CoA approximately myristoyl-CoA > stearoyl-CoA > oleoyl-CoA approximately = arachidonoyl-CoA) present either as monomers in solution or guests in host membrane bilayers. Site-directed mutagenesis of the iPLA2beta catalytic serine (S465A) completely abolished acyl-CoA thioesterase activity, demonstrating that Ser-465 catalyzes both phospholipid and acyl-CoA hydrolysis. Remarkably, incubation of iPLA2beta with oleoyl-CoA, but not other long-chain acyl-CoAs, resulted in robust stoichiometric covalent acylation of the enzyme. Moreover, S465A mutagenesis or pretreatment of wild-type iPLA2beta with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one unexpectedly increased acylation of the enzyme, indicating the presence of a second reactive nucleophilic residue that participates in the formation of the fatty acyl-iPLA2beta adduct. Radiolabeling of intact Sf9 cells expressing iPLA2beta with [3H]oleic acid demonstrated oleoylation of the membrane-associated enzyme. Partial trypsinolysis of oleoylated iPLA2beta and matrix-assisted laser desorption ionization mass spectrometry analysis localized the acylation site to a hydrophobic 25-kDa fragment (residues approximately 400-600) spanning the active site to the calmodulin binding domain. Intriguingly, calmodulin-Ca2+ blocked acylation of iPLA2beta by oleoyl-CoA. Remarkably, the addition of low micromolar concentrations (5 microM) of oleoyl-CoA resulted in reversal of calmodulin-mediated inhibition of iPLA2 beta phospholipase A2 activity. These results collectively identify the molecular species-specific acyl-CoA thioesterase activity of iPLA2beta, demonstrate the presence of a second active site that mediates iPLA2beta autoacylation, and identify long-chain acyl-CoAs as potential candidates mediating calcium influx factor activity.     

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH.     

Effect of modification of the length and flexibility of the acyl carrier protein-thioesterase interdomain linker on functionality of the animal fatty acid synthase

A natural linker of approximately 20 residues connects the acyl carrier protein with the carboxy-terminal thioesterase domain of the animal fatty acid synthase. This study examines the effects of changes in the length and amino acid composition of this linker on catalytic activity, product composition, and segmental motion of the thioesterase domain. Deletion of 10 residues, almost half of the interdomain linker, had no effect on either mobility of the thioesterase domain, estimated from fluorescence polarization of a pyrenebutyl methylphosphono moiety bound covalently to the active site serine residue, or functionality of the fatty acid synthase; further shortening of the linker limited mobility of the thioesterase domain and resulted in reduced fatty acid synthase activity and an increase in product chain length from 16 to 18 and 20 carbon atoms. Surprisingly, however, even when the entire linker region was deleted, the fatty acid synthase retained 28% activity. Lengthening of the linker, by insertion of an unusually long acyl carrier protein-thioesterase linker from a modular polyketide synthase, increased mobility of the thioesterase domain without having any significant effect on catalytic properties of the complex. Interdomain linkers could also be used to tether, to the acyl carrier protein domain of the fatty acid synthase, a thioesterase active toward shorter chain length acyl thioesters generating novel short-chain fatty acid synthases. These studies reveal that although truncation of the interdomain linker partially impacts the ability of the thioesterase domain to terminate growth of the acyl chain, the overall integrity of the fatty acid synthase is quite tolerant to moderate changes in linker length and flexibility. The retention of fatty acid synthesizing activity on deletion of the entire linker region implies that the inherent flexibility of the phosphopantetheine "swinging arm" also contributes significantly to the successful docking of the long-chain acyl moiety in the thioesterase active site.     

Conserved residues in the putative catalytic triad of human bile acid Coenzyme A:amino acid N-acyltransferase

Human bile acid-CoA:amino acid N-acyltransferase (hBAT), an enzyme catalyzing the conjugation of bile acids with the amino acids glycine or taurine has significant sequence homology with dienelactone hydrolases and other alpha/beta hydrolases. These enzymes have a conserved catalytic triad that maps onto the mammalian BATs at residues Cys-235, Asp-328, and His-362 of the human sequence, albeit that the hydrolases contain a serine instead of a cysteine. In the present study, the function of the putative catalytic triad of hBAT was examined by chemical modification with the cysteine alkylating reagent N-ethylmaleimide (NEM) and by site-directed mutagenesis of the triad residues followed by enzymology studies of mutant and wild-type hBATs. Treatment with NEM caused inactivation of wild-type hBAT. However, preincubation of wild-type hBAT with the substrate cholyl-CoA before NEM treatment prevented loss of N-acyltransferase activity. Substitution of His-362 or Asp-328 with alanine results in inactivation of hBAT. Although substitution of Cys-235 with serine generated an hBAT mutant with lower N-acyltransferase activity, it substantially increased the bile acid-CoA thioesterase activity compared with wild type. In summary, data from this study support the existence of an essential catalytic triad within hBAT consisting of Cys-235, His-362, and Asp-328 with Cys-235 serving as the probable nucleophile and thus the site of covalent attachment of the bile acid molecule.     

Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase

Each of the three cysteinyl residues per subunit in D-amino acid transaminase from a thermophilic species of Bacillus has been changed to a glycine residue (C142G, C164G, and C212G) by site-directed mutagenesis. The mutant enzymes were detected by Western blots and a stain for activity. After purification to homogeneity, each mutant protein had the same activity as the wild-type enzyme. Thus, none of the Cys residues are essential for catalysis. Each protein when denatured showed the expected titer of two SH groups per subunit. In the native state, each of the three mutant proteins exhibited nearly the same slow rate of titration of SH groups as the wild-type protein with about one SH group titratable over a period of 4 h. Conversion of Ser-146, adjacent to Lys-145 to which the coenzyme pyridoxal phosphate is bound, to an alanine residue (S146A) does not alter the catalytic activity but has a significant effect on the SH titration behavior. Thus, three to four of the six SH groups of S146A are titratable by DTNB. The rapid SH titration of S146A is prevented by the presence of D-alanine. This finding suggests that the change of Ser-146 to Ala at the active site promotes the exposure and rapid titration of a Cys residue in that region. The rapid SH titration of S146A by DTNB is accompanied by a loss of enzyme activity. Two of the mutant enzymes, C142G and S146A, lose activity at 4 degrees C and also upon freezing and thawing. The mutant enzymes C164G and C212G show the same degree of thermostability as the wild-type enzyme.     

Acylpeptide hydrolase: inhibitors and some active site residues of the human enzyme.

Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions.     

Active-site mutants of beta-lactamase: use of an inactive double mutant to study requirements for catalysis

We have studied the catalytic activity and some other properties of mutants of Escherichia coli plasmid-encoded RTEM beta-lactamase (EC 3.5.2.6) with all combinations of serine and threonine residues at the active-site positions 70 and 71. (All natural beta-lactamases have conserved serine-70 and threonine-71.) From the inactive double mutant Ser-70----Thr, Thr-71----Ser [Dalbadie-McFarland, G., Cohen, L. W., Riggs, A. D., Morin, C., Itakura, K., & Richards, J. H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6409-6413], an active revertant, Thr-71----Ser (i.e., residue 70 in the double mutant had changed from threonine to the serine conserved at position 70 in the wild-type enzyme), was isolated by an approach that allows identification of active revertants in the absence of a background of wild-type enzyme. This mutant (Thr-71----Ser) has about 15% of the catalytic activity of wild-type beta-lactamase. The other possible mutant involving serine and threonine residues at positions 70 and 71 (Ser-70----Thr) shows no catalytic activity. The primary nucleophiles of a serine or a cysteine residue [Sigal, I. S., Harwood, B. G., & Arentzen, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] at position 70 thus seem essential for enzymatic activity. Compared to wild-type enzyme, all three mutants show significantly reduced resistance to proteolysis; for the active revertant (Thr-71----Ser), we have also observed reduced thermal stability and reduced resistance to denaturation by urea.     

Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its Mechanism

O-Linked glycosylation is one of the most abundant post-translational modifications of proteins. Within the secretory pathway of higher eukaryotes, the core of these glycans is frequently an N-acetylgalactosamine residue that is α-linked to serine or threonine residues. Glycoside hydrolases in family 101 are presently the only known enzymes to be able to hydrolyze this glycosidic linkage. Here we determine the high-resolution structures of the catalytic domain comprising a fragment of GH101 from Streptococcus pneumoniae TIGR4, SpGH101, in the absence of carbohydrate, and in complex with reaction products, inhibitor, and substrate analogues. Upon substrate binding, a tryptophan lid (residues 724-WNW-726) closes on the substrate. The closing of this lid fully engages the substrate in the active site with Asp-764 positioned directly beneath C1 of the sugar residue bound within the −1 subsite, consistent with its proposed role as the catalytic nucleophile. In all of the bound forms of the enzyme, however, the proposed catalytic acid/base residue was found to be too distant from the glycosidic oxygen (>4.3 Å) to serve directly as a general catalytic acid/base residue and thereby facilitate cleavage of the glycosidic bond. These same complexes, however, revealed a structurally conserved water molecule positioned between the catalytic acid/base and the glycosidic oxygen. On the basis of these structural observations we propose a new variation of the retaining glycoside hydrolase mechanism wherein the intervening water molecule enables a Grotthuss proton shuttle between Glu-796 and the glycosidic oxygen, permitting this residue to serve as the general acid/base catalytic residue.     

Role of ser-237 in the substrate specificity of the carbapenem-hydrolyzing class A beta-lactamase Sme-1.

The role of the serine residue found at position 237 in the carbapenemase Sme-1 has been investigated by constructing a mutant in which Ser-237 was replaced by an alanine. The S237A mutant showed a catalytic behavior against penicillins and aztreonam very similar to that of Sme-1. By contrast, S237A was characterized by a reduced catalytic efficiency against cephems, such as cephalothin and cephaloridine. In addition, the weak activity of Sme-1 against the cephamycin cefoxitin was hardly detectable with the mutant enzyme. Finally, the Ser-237-->Ala mutation resulted in a marked decrease in catalytic activity against imipenem, showing that Ser-237 contributes to the carbapenemase activity of the class A beta-lactamase Sme-1.     

The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O_δ2–C_γ bond appears to be a double bond, with O_δ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O_δ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF_obs − DF_calc density above 2.5 σ next to O_δ1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK_a of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK_a histidine theory.     

Site-directed mutagenesis of active-site-related residues in Torpedo acetylcholinesterase. Presence of a glutamic acid in the catalytic triad.

Site-directed mutagenesis was used to investigate the role of acidic amino acid residues close to the active site of Torpedo acetylcholinesterase. The recently determined atomic structure of this enzyme shows the conserved Glu-327, together with His-440 and Ser-200 as forming a catalytic triad, while the adjacent conserved Asp-326 points away from the active site. Transfection of appropriately mutated DNA into COS cells showed that the mutation of Asp-326----Asn had little effect on catalytic activity or the molecular forms expressed, suggesting no crucial structural or functional role for this residue. Mutation of Glu-327 to Gln or to Asp led to an inactive product. These results support the conclusions of the structural analysis for the two acidic residues.     

Conformational mobility of His-64 in the Thr-200----Ser mutant of human carbonic anhydrase II

The three-dimensional structure of the Thr-200----Ser (T200S) mutant of human carbonic anhydrase II (CAII) has been determined by X-ray crystallographic methods at 2.1-A resolution. This particular mutant of CAII exhibits CO2 hydrase activity that is comparable to that of the wild-type enzyme with a 2-fold stabilization of the E.HCO3- complex and esterase activity that is 4-fold greater than that of the wild-type enzyme. The structure of the mutant enzyme reveals no significant local changes accompanying the conservative T200S substitution, but an important nonlocal structural change is evident: the side chain of catalytic residue His-64 rotates away from the active site by 105 degrees about chi 1 and apparently displaces a water molecule. The displaced water molecule is present in the wild-type enzyme; however, the electron density into which this water is built is interpretable as an alternate conformation of His-64 with 10-20% occupancy. The rate constants for proton transfer from the zinc-water ligand to His-64 and from His-64 to bulk solvent are maintained in the T200S variant; therefore, if His-64 is conformationally mobile about chi 1 and/or chi 2 during catalysis, compensatory changes in solvent configuration must sustain efficient proton transfer.     

Identification of putative active site residues of ACAT enzymes

In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.     

Complete amino acid sequence of the thioesterase domain of chicken liver fatty acid synthase

The complete amino acid sequence of thioesterase domain of chicken liver fatty acid synthase has been determined by sequencing peptides produced by trypsin, Staphylococcus aureus V8 protease, and cyanogen bromide cleavage. The thioesterase domain consists of 300 amino acid residues. All of the tryptic peptides of the thioesterase domain were isolated and sequenced, except the segment covered from position 109 to position 124. Peptides resulting from digestion by Staphylococcus aureus V8 protease and cyanogen bromide cleavage filled the missing part and overlapped the complete sequence of the entire thioesterase domain. The NH2 terminus of the thioesterase domain was determined to be lysine by sequencing the whole domain up to 20 residues while the COOH terminus was identified as serine through carboxyl peptidase Y cleavage. The active site of the thioesterase domain of chicken fatty acid synthase was suggested to be the serine on position 101 according to its homology with other serine-type esterases and proteases which have a common structure of -Gly-X-Ser-Y-Gly- with the variable amino acids X and Y disrupting the homology.