An Acyl-covalent Enzyme Intermediate of Lecithin:Retinol Acyltransferase

Synthesis of fatty acid retinyl esters determines systemic vitamin A levels and provides substrate for production of visual chromophore (11-cis-retinal) in vertebrates. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to retinol. To delineate the catalytic mechanism of this reaction, we expressed and purified a fully active, soluble form of this enzyme and used it to examine the possible formation of a transient acyl-enzyme intermediate. Detailed mass spectrometry analyses revealed that LRAT undergoes spontaneous, covalent modification upon incubation with a variety of phosphatidylcholine substrates. The addition of an acyl chain occurs at the Cys¹⁶¹ residue, indicating formation of a thioester intermediate. This observation provides the first direct experimental evidence of thioester intermediate formation that constitutes the initial step in the proposed LRAT catalytic reaction. Additionally, we examined the effect of increasing fatty acyl side chain length in phosphatidylcholine on substrate accessibility in this reaction, which provided insights into the function of the single membrane-spanning domain of LRAT. These observations are critical to understanding the catalytic mechanism of LRAT protein family members as well as other lecithin:acyltransferases wherein Cys residues are required for catalysis.     

Residues located on membrane‐embedded flexible loops are essential for the second step of the apolipoprotein N‐acyltransferase reaction

Apolipoprotein N‐acyltransferase (Lnt) is an essential membrane‐bound enzyme that catalyzes the third and last step in the post‐translational modification of bacterial lipoproteins. In order to identify essential residues implicated in substrate recognition and/or binding we screened for non‐functional variants of Lnt obtained by error‐prone polymerase chain reaction in a complementation assay using a lnt depletion strain. Mutations included amino acid substitutions in the active site and of residues located on flexible loops in the catalytic periplasmic domain. All, but one mutation, led to the formation of the thioester acyl‐enzyme intermediate and to the accumulation of apo‐Lpp, suggesting that these residues are involved in the second step of the reaction. A large cytoplasmic loop contains a highly conserved region and two hydrophobic segments. Accessibility analysis to alkylating reagents of substituted cysteine residues introduced in this region demonstrated that the hydrophobic segments do not completely span the membrane. Two residues in the highly conserved cytoplasmic region were shown to be essential for Lnt function. Together, our data suggest that amino acids located on flexible cytoplasmic and periplasmic loops, predicted to be membrane embedded, are required for efficient N‐acylation of lipoproteins.     

Aminoacylation of the N-terminal cysteine is essential for Lol-dependent release of lipoproteins from membranes but does not depend on lipoprotein sorting signals

Lipoproteins are present in a wide variety of bacteria and are anchored to membranes through lipids attached to the N-terminal cysteine. The Lol system of Escherichia coli mediates the membrane-specific localization of lipoproteins. Aspartate at position 2 functions as a Lol avoidance signal and causes the retention of lipoproteins in the inner membrane, whereas lipoproteins having residues other than aspartate at position 2 are released from the inner membrane and localized to the outer membrane by the Lol system. Phospholipid:apolipoprotein transacylase, Lnt, catalyzes the last step of lipoprotein modification, converting apolipoprotein into mature lipoprotein. To reveal the importance of this aminoacylation for the Lol-dependent membrane localization, apolipoproteins were prepared by inhibiting lipoprotein maturation. Lnt was also purified and used to convert apolipoprotein into mature lipoprotein in vitro. The release of these lipoproteins was examined in proteoliposomes. We show here that the aminoacylation is essential for the Lol-dependent release of lipoproteins from membranes. Furthermore, lipoproteins with aspartate at position 2 were found to be aminoacylated both in vivo and in vitro, indicating that the lipoprotein-sorting signal does not affect lipid modification.     

Overexpression of LolCDE allows deletion of the Escherichia coli gene encoding apolipoprotein N-acyltransferase

Bacterial lipoproteins represent a subset of membrane-associated proteins that are covalently modified with lipids at the N-terminal cysteine. The final step of lipoprotein modification, N-acylation of apolipoproteins, is mediated by apolipoprotein N-acyltransferase (Lnt). Examinations with reconstituted proteoliposomes and a conditional mutant previously indicated that N-acylation of lipoproteins is required for their efficient release from the inner membrane catalyzed by LolA and LolCDE, the lipoprotein-specific chaperone and ABC transporter, respectively. Because Lnt is essential for Escherichia coli, a mutant lacking Lnt activity has not been isolated. However, we report here that lnt-null strains can be constructed when LolCDE is overproduced in strains lacking either the major outer membrane lipoprotein Lpp or transpeptidases that cross-link Lpp with peptidoglycan. Lipoproteins purified from the lnt-null strain exhibited increased mobility on SDS-PAGE compared to those from wild-type cells and could be sequenced by Edman degradation, indicating that lipoproteins in this mutant exist as apolipoproteins that lack N-acylation. Overexpression of Lpp in the lnt-null strain resulted in the accumulation of apoLpp in the inner membrane and caused growth arrest. In contrast to the release of mature Lpp in the presence of LolA and LolCDE, that of apoLpp from the inner membrane was significantly retarded. Furthermore, the amount of lipoproteins copurified with LolCDE was significantly reduced in the lnt-null strain. These results indicate that the affinity of LolCDE for apolipoprotein is very low, and therefore, overexpression of LolCDE is required for its release and sorting to the outer membrane.     

Fatty acyl chain specificity of phosphatidylcholine hydrolysis catalyzed by lipoprotein lipase. Effect of apolipoprotein C-II and its (56-79) synthetic fragment

Mixed acyl chain phosphatidylcholine molecules in Triton N-101 micelles were employed as substrates for lipoprotein lipase to test which substrate acyl chain has the greatest effect on activation of the enzyme by apolipoprotein C-II. The phospholipase A1 activity of lipoprotein lipase was measured by pH-stat. The activation factor (lipoprotein lipase activity plus apolipoprotein C-II/activity minus apolipoprotein C-II) increased monotonically with apolipoprotein C-II concentration up to 1 microM apolipoprotein C-II at an enzyme concentration of 0.01 microM. The maximal activation factor for phosphatidylcholine substrate molecules with sn-2 acyl chain lengths of 14 averages 14.8. By contrast, for sn-2 acyl chain lengths of 16 the activation factor was 29.2. Varying the sn-1 acyl chain length had no significant effect on the activation factor. The chain-length dependence of the activation factor is similar with the apolipoprotein C-II peptide fragment comprising residues 56-79, which does not include the lipid-binding region of apolipoprotein C-II. These data are consistent with a model for activation of lipoprotein lipase in which residues 56-79 bind to lipoprotein lipase and alter the interaction of the sn-2 acyl chain of the phosphatidylcholine (PC) substrate or the lysoPC product within the activated state complex.     

Phosphatidylethanolamine is not essential for the N-acylation of apolipoprotein in Escherichia coli

It has been postulated that the N-acyl fatty acid attached to the amino terminus of the major Escherichia coli lipoprotein is derived from the fatty acid at the 1-position of phosphatidylethanolamine (PtdEtn) (Jackowski, S., and Rock, C.O. (1986) J. Biol. Chem. 261, 11328-11333). To ascertain the role of PtdEtn in the conversion of apolipoprotein to the mature lipoprotein, the lipoprotein from E. coli strain AH930 (pss::kan) containing a null mutation in the phosphatidylserine synthase gene (pss) was studied. Pulse labeling with [35S]methionine for 30 s or 5 min revealed the formation of mature lipoprotein in both wild-type (W3110) and mutant (AH930) cells. [3H]Palmitate-labeled lipoproteins from both the mutant and wild-type cells were found to contain nearly identical amounts of alkali-resistant (amide-linked, 41-42%) and alkali-labile (ester-linked, 58-59%) fatty acids. Edman degradation and dansylation of the immuno-affinity-purified [35S]cysteine-labeled lipoprotein showed that the NH2 terminus of the lipoprotein in the mutant was blocked as in the wild type. In vitro assay of apolipoprotein N-acyltransferase using membranes either from the mutant or the wild-type strain as the source of both the enzyme and the acyl donor revealed that both membranes were equally active in the conversion of [35S]methionine-labeled apolipoprotein to lipoprotein. These data strongly suggest that PtdEtn is not essential for the N-acylation of apolipoprotein to form lipoprotein, and other major phospholipids such as phosphatidylglycerol and cardiolipin can serve as the donor of fatty acid in the N-acylation of apolipoprotein.     

Depletion of apolipoprotein N-acyltransferase causes mislocalization of outer membrane lipoproteins in Escherichia coli

Lipoproteins in Gram-negative Enterobacteriaceae carry three fatty acids on the N-terminal cysteine residue, two as a diacylglyceride and one through an N-linkage following signal peptide cleavage. Most lipoproteins are anchored in the outer membrane, facing the periplasm, but some lipoproteins remain in the plasma membrane, depending on the amino acid at position +2, immediately after the fatty-acylated cysteine. In vitro, the last step in lipoprotein maturation, N-acylation of apolipoproteins by the plasma membrane apolipoprotein N-acyltransferase (Lnt), is necessary for efficient recognition of outer membrane lipoproteins by the Lol system, which transports them from the plasma to the outer membrane (Fukuda, A., Matsuyama, S.-I., Hara, T., Nakayama, J., Nagasawa, H., and Tokuda, H. (2002) J. Biol. Chem. 277, 43512-43518). To study the role of Lnt in vivo, we constructed a conditional lnt mutant of Escherichia coli. The apo-form of peptidoglycan-anchored major lipoprotein (Lpp) and two other outer membrane lipoproteins accumulated in the plasma membrane when lnt expression was reduced. We also found that Lnt is an essential protein in E. coli and that the lethality is partially because of the retention of apoLpp in the plasma membrane. Topology mapping of Lnt with beta-galactosidase and alkaline phosphatase fusions indicated the presence of six membrane-spanning segments. The lnt gene in a mutant of Salmonella enterica displaying thermosensitive Lnt activity (Gupta, S. D., Gan, K., Schmid, M. B., and Wu, H. C. (1993) J. Biol. Chem. 268, 16551-16556) was found to carry a mutation causing a single glutamate to lysine substitution at a highly conserved position in the last predicted periplasmic loop of the protein.     

Inhibition of mammalian fatty acid synthetase activity by NADP involves decreased mobility of the 4'-phosphopantetheine prosthetic group

Mammalian fatty acid synthetase carrying a 3-keto, 3-hydroxy, or 2-enoyl acyl-enzyme intermediate on the 4'-phosphopantetheine thiol is reversibly inhibited by binding of NADP to the enoyl reductase domain. Acyl moieties which can normally leave the enzyme by thioester hydrolysis or by transfer to a CoA acceptor cannot readily be removed from the NADP-inhibited enzyme; in addition, 3-keto or 2-enoyl moieties attached to the enzyme 4'-phosphopantetheine cannot readily be reduced when NADP is replaced by NADPH, even though model substrates can be reduced immediately. Reactivation of the NADP-inhibited 3-ketoacyl-enzyme, by exposure to NADPH, is paralleled by reduction and dehydration of the 3-ketoacyl moiety to a saturated acyl moiety without accumulation of either the 3-hydroxy or 2-enoyl acyl-enzyme intermediates, indicating that once the 4'-phosphopantetheine engages the ketoacyl moiety in the ketoreductase domain, subsequent reactions occur very rapidly. The results are consistent with a hypothesis which proposes that NADP binding to the enoyl reductase domain of fatty acid synthetase carrying an acyl intermediate other than a saturated moiety induces a conformational change in the enzyme that results in decreased mobility of the 4'-phosphopantetheine prosthetic group. Normal mobility of the prosthetic group, essential for transfer of acyl-enzyme intermediates through the active sites of the various functional domains, is restored relatively slowly when NADP is replaced by NADPH. It remains to be determined whether this modulation by pyridine nucleotides observed in vitro plays a role in the regulation of fatty acid synthetase activity in vivo.     

Identification of essential residues in apolipoprotein N-acyl transferase, a member of the CN hydrolase family

Apolipoprotein N-acyl transferase (Lnt) is an essential membrane-bound protein involved in lipid modification of all lipoproteins in gram-negative bacteria. Essential residues in Lnt of Escherichia coli were identified by using site-directed mutagenesis and an in vivo complementation assay. Based on sequence conservation and known protein structures, we predict a model for Lnt, which is a member of the CN hydrolase family. Besides the potential catalytic triad E267-K335-C387, four residues that directly affect the modification of Braun's lipoprotein Lpp are absolutely required for Lnt function. Residues Y388 and E389 are part of the hydrophobic pocket that constitutes the active site. Residues W237 and E343 are located on two flexible arms that face away from the active site and are expected to open and close upon the binding and release of phospholipid and/or apolipoprotein. Substitutions causing temperature-dependent effects were located at different positions in the structural model. These mutants were not affected in protein stability. Lnt proteins from other proteobacteria, but not from actinomycetes, were functional in vivo, and the essential residues identified in Lnt of E. coli are conserved in these proteins.     

Actinorhodin biosynthesis: structural requirements for post-PKS tailoring intermediates revealed by functional analysis of ActVI-ORF1 reductase

Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) is an aromatic polyketide antibiotic, whose basic carbon skeleton is derived from type II polyketide synthase (PKS). Although an acyl carrier protein (ACP) serves as an anchor of nascent intermediates during chain elongation in the type II PKS complex, it generally remains unknown when an ACP-free intermediate is released from the complex to post-PKS modification ("tailoring") steps. In ACT biosynthesis, a stereospecific ketoreductase (RED1) encoded by actVI-ORF1 reduces the 3beta-keto group of a proposed bicyclic intermediate to an (S) secondary alcohol. The bicyclic intermediate is formed from the steps of PKS and its closely associated enzymes and lies at the interface toward ACT-tailoring steps. To clarify whether RED1 recognizes the ACP-bound bicyclic intermediate or the ACP-free bicyclic intermediate, recombinant RED1 was purified for enzymatic characterization. RED1 was heterologously expressed in Escherichia coli and purified using Ni-chelate and gel filtration column chromatographies to homogeneity in soluble form. Enzymatic studies in vitro on RED1 with synthetic analogues, in place of an unstable bicyclic intermediate, showed that RED1 recognizes 3-oxo-4-naphthylbutyric acid (ONBA) as a preferred substrate and not its N-acetylcysteamine thioester. This strongly suggests that RED1 recognizes ACP-free bicyclic beta-keto acid as the first committed intermediate of tailoring steps. Kinetic studies of RED1 showed high affinity with ONBA, consistent with the requirement for an efficient reduction of a labile beta-keto carboxylic acid. Interestingly, the methyl ester of ONBA acted as a competitive inhibitor of RED1, indicating the presence of strict substrate recognition toward the terminal acid functionality.     

The thioesterase domain from the pimaricin and erythromycin biosynthetic pathways can catalyze hydrolysis of simple thioester substrates

The recombinant polyketide synthase thioesterase domains from the pimaricin and 6-deoxyerythronolide B biosynthetic pathways catalyze hydrolysis of a number of simple N-acetylcysteamine thioester derivatives. This study demonstrates that thioesterases are not highly substrate selective in formation of the acyl-enzyme intermediate, in contrast to non-ribosomal peptide synthase thioesterase domains that show very high specificity for substrate loading. This observation has important implications for the engineering of biosynthetic pathways to produce polyketide products.     

The carboxyl-terminal region of thioesterase II participates in the interaction with fatty acid synthase. Use of electrospray ionization mass spectrometry to identify a carboxyl-terminally truncated form of the enzyme

Medium-chain S-acyl fatty acid synthase thioester hydrolase (thioesterase II), a discrete 263-residue serine active-site enzyme, modifies the product specificity of the de novo lipogenic pathway in certain specialized tissues by hydrolyzing the thioester bond linking the growing acyl chain to the 4'-phosphopantetheine of the fatty acid synthase. Modification of one thioesterase II cysteine thiol with thionitrobenzoate inhibited interaction with the S-acyl-fatty acid synthase substrate but not with acyl-CoA model substrates. The identity of the sensitive cysteine residue was determined by treatment of the thionitrobenzoyl enzyme with cyanide and cleavage at the amino-terminal side of the S-cyanocysteinyl residue. Two small cleavage products were isolated; their molecular masses (889 and 675 Da) and amino acid compositions indicated that both originated from cleavage at Cys256. A new technique of electrospray ionization mass spectrometry was utilized to confirm that the heterogeneity displayed by the products of S-cyanocysteinyl cleavage resulted from the presence, in the purified preparations, of both full-length and a truncated form of the enzyme missing the carboxyl-terminal Leu-Thr peptide. The proportion of full-length polypeptide present appeared to correlate with the activity of the enzyme toward its natural substrate. The results of modification of Cys256 by thionitrobenzoate and removal of residues 262 and 263 by endogenous proteases indicate that integrity of the carboxyl-terminal region is important for interaction with its acyl-fatty acid synthase substrate.     

Kinetic characterization of long chain fatty acyl coenzyme A ligase from rat liver mitochondria.

Long chain fatty acyl coenzyme A ligase (EC 6.2.1.3) purified from rat liver mitochondria has been characterized with respect to several kinetic parameters. Many of the kinetic properties of the mitochondrial enzyme are similar to those of the purified microsomal enzyme with respect to palmitoyl-CoA formation, but there are distinct differences. The fatty acid and nucleotide specificities of the mitochondrial enzyme are similar to those of the microsomal enzyme, as are the apparent Km values for ATP and coenzyme A. On the other hand, the mitochondrial enzyme differs from the microsomal enzyme in that it has a lower pH optimum, is different in molecular weight, and does not show simple saturation kinetics with palmitate as substrate. Of particular interest is the evidence presented which indicates that the mitochondrial long chain fatty acyl-CoA ligase, unlike short and medium chain ligases, does not utilize an acyladenylate as an intermediate in the formation of fatty acyl-CoA.     

A radiochemical assay for glycine N-acyltransferase activity. Some properties of the enzyme in rat and rabbit.

We have developed a sensitive radiochemical assay of glycine N-acyltransferase activity, using phenylacetyl-CoA as the acyl donor and glycine as the acceptor. This assay measures formation of the product, phenylacetylglycine, instead of disappearance of the substrate, phenylacetyl-CoA, as did earlier assays. The subcellular location and some properties of the conjugating activity were determined in liver and kidney of the rabbit and the rat. Rabbit lung and intestine were also tested for activity.     

Synthesis of long chain acyl-enzyme thioesters by modified fatty acid synthetases and their hydrolysis by a mammary gland thioesterase.

The fatty acid synthetase multienzyme from lactating rat mammary gland was modified either by removal of the two thioesterase I domains with trypsin or by inhibiting the thioesterase I activity with phenylmethanesulfonyl fluoride. The modified multienzymes are able to convert acetyl-CoA, malonyl-CoA, and NADPH to long chain acyl moieties (C₁₆C₂₂), which are covalently bound to the enzyme through thioester linkage, but they are unable to release the acyl groups as free fatty acids. A single enzyme-bound, long chain acyl thioester is formed by each molecule of modified multienzyme. Kinetic studies showed that the modified multienzymes rapidly elongate the acetyl primer moiety to a C₁₆ thioester and that further elongation to C₁₈, C₂₀, and C₂₂ is progressively slower. Thioesterase II, a mammary gland enzyme which is not part of the fatty acid synthetase multienzyme, can release the acyl moiety from its thioester linkage to either modified multienzyme. Kinetic data are consistent with the formation of an enzyme—substrate complex between thioesterase II and the acylated modified multienzymes. The present study demonstrates that the ability of thioesterase II to modify the product specificity of normal fatty acid synthetase is most likely attributable to the capacity of thioesterase II for hydrolysis of acyl moieties from thioester linkage to the multienzyme.     

Resonance Raman evidence for substrate reorginization in the active site of papain.

Resonance Raman spectra were obtained for the acylenzyme 4-dimethylamino-3-nitro(alpha-benzamido)cinnamoyl-papain prepared using the chromophoric substrate methyl 4-dimethylamino-3-nitro(alpha-benzamido)cinnamate. These spectra contained vibrational spectral data of the acyl residue while covalently attached to the active site and could be used to follow directly acylation and deacylation kinetics. Spectra were obtained at pH values ranging from those where the acyl-enzyme is relatively stable (pH 3.0, tau 1/2 congruent to 800 s) to those where it is relatively unstable (pH 9.2, tau 1/2 congruent to 223 s). Throughout this range acyl-enzyme spectra differed completely from that of the free substrate or the product (4-dimethylamino-3-nitro(alpha-benzamido)cinnamic acid) indicating that a structural change occurred on combination with the active site. The spectra are consistent with rearrangement of the alpha-benzamido group in the bound substrate, -NH--C(==O)Ph becoming --N==C(--OX)Ph, where the bonding to oxygen is unknown. Superimposed on these large differences, small changes in acyl-enzyme spectra also occurred as pH was raised to decrease the half-life. All of the above spectral perturbations are consistent with a structural change in the acyl-enzyme which precedes the rate-determining step in deacylation. Thus, deacylation proceeds from an acyl residue structure differing from that of the substrate in solution. Upon acid denaturation the spectrum characteristic of the intermediate reverts to one closely resembling the substrate, demonstrating that a functioning active site is necessary to produce the observed differences. Spectra in D2O of native acyl-enzyme were identical with those in H2O, indicating that the observed differences in rate constant were not due to solvent-induced structural changes. Activated papain purified by crystallization or by affinity chromatography formed the acyl-enzyme. However, the kinetics of formation and deacylation differed between these materials, as did the spectral properties. Small differences in active-site structure are considered to be responsible for this effect, and it is suggested that such spectral perturbations may be useful in directly relating small differences in structure of the substrate in the active site with corresponding differences in kinetics.     

Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl substrate.

Enoyl-ACP reductases participate in fatty acid biosynthesis by utilizing NADH to reduce the trans double bond between positions C2 and C3 of a fatty acyl chain linked to the acyl carrier protein. The enoyl-ACP reductase from Mycobacterium tuberculosis, known as InhA, is a member of an unusual FAS-II system that prefers longer chain fatty acyl substrates for the purpose of synthesizing mycolic acids, a major component of mycobacterial cell walls. The crystal structure of InhA in complex with NAD+ and a C16 fatty acyl substrate, trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester, reveals that the substrate binds in a general "U-shaped" conformation, with the trans double bond positioned directly adjacent to the nicotinamide ring of NAD+. The side chain of Tyr158 directly interacts with the thioester carbonyl oxygen of the C16 fatty acyl substrate and therefore could help stabilize the enolate intermediate, proposed to form during substrate catalysis. Hydrophobic residues, primarily from the substrate binding loop (residues 196-219), engulf the fatty acyl chain portion of the substrate. The substrate binding loop of InhA is longer than that of other enoyl-ACP reductases and creates a deeper substrate binding crevice, consistent with the ability of InhA to recognize longer chain fatty acyl substrates.     

Further kinetic and molecular characterization of an extremely heat-stable carboxylesterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

The carboxylesterase (serine esterase, EC 3.1.1.1) from Sulfolobus acidocaldarius was purified 940-fold to homogeneity by an improved purification procedure with a yield of 57%. In the presence of alcohols the enzyme catalyses the transfer of the substrate acyl group to alcohols in parallel to hydrolysis. The results show the existence of an alcohol-binding site and a competitive partitioning of the acyl-enzyme intermediate between water and alcohols. Aniline acts also as a nucleophilic acceptor for the acyl group. On the basis of titration with diethyl p-nitrophenyl phosphate, a number of four active centres is determined for the tetrameric carboxylesterase. The sequence of 20 amino acid residues at the esterase N-terminus and the amino acid composition are reported.     

DHHC protein S-acyltransferases use similar ping-pong kinetic mechanisms but display different acyl-CoA specificities

DHHC proteins catalyze the reversible S-acylation of proteins at cysteine residues, a modification important for regulating protein localization, stability, and activity. However, little is known about the kinetic mechanism of DHHC proteins. A high-performance liquid chromatography (HPLC), fluorescent peptide-based assay for protein S-acylation activity was developed to characterize mammalian DHHC2 and DHHC3. Time courses and substrate saturation curves allowed the determination of V(max) and K(m) values for both the peptide N-myristoylated-GCG and palmitoyl-coenzyme A. DHHC proteins acylate themselves upon incubation with palmitoyl-CoA, which is hypothesized to reflect a transient acyl enzyme transfer intermediate. Single turnover assays with DHHC2 and DHHC3 demonstrated that a radiolabeled acyl group on the enzyme transferred to the protein substrate, consistent with a two-step ping-pong mechanism. Enzyme autoacylation and acyltransfer to substrate displayed the same acyl-CoA specificities, further supporting a two-step mechanism. Interestingly, DHHC2 efficiently transferred acyl chains 14 carbons and longer, whereas DHHC3 activity was greatly reduced by acyl-CoAs with chain lengths longer than 16 carbons. The rate and extent of autoacylation of DHHC3, as well as the rate of acyl chain transfer to protein substrate, were reduced with stearoyl-CoA when compared with palmitoyl-CoA. This is the first observation of lipid substrate specificity among DHHC proteins and may account for the differential S-acylation of proteins observed in cells.     

Spectral observation of an acyl-enzyme intermediate of lipoprotein lipase

There have been several studies indicating that hydrolysis reactions of fatty acid esters catalyzed by lipases proceed through an acyl-enzyme intermediate typical of serine proteases. In particular, one careful kinetic study with the physiologically important enzyme lipoprotein lipase (LPL) is consistent with rate-limiting deacylation of such an intermediate. To observe the spectrum of acyl-enzyme and study the mechanism of LPL-catalyzed hydrolysis of substrate, we have used a variety of furylacryloyl substrates including 1,2-dipalmitoyl-3-[(beta-2-furylacryloyl)triacyl]glyceride (DPFATG) to study the intermediates formed during the hydrolysis reaction catalyzed by the enzyme. After isolation and characterization of the molecular weight of adipose LPL, we determined its extinction coefficient at 280 nm to quantitate the formation of any acyl-enzyme intermediate formed during substrate hydrolysis. We observed an intermediate at low pH during the enzyme-catalyzed hydrolysis of (furylacryloyl)imidazole. This intermediate builds early in the reaction when a substantial amount of substrate has hydrolyzed but no product, furylacrylate, has been formed. The acyl-enzyme has a lambda max = 305 nm and a molar extinction coefficient of 22,600 M-1 cm-1; these parameters are similar to those for furylacryloyl esters including the serine ester. These data provide the first spectral evidence for a serine acyl-enzyme in lipase-catalyzed reactions. The LPL hydrolysis reaction is base catalyzed, exhibiting two pKa values; the more acidic of these is 6.5, consistent with base catalysis by histidine. The biphasic rates for substrate disappearance or product appearance and the absence of leaving group effect indicate that deacylation of intermediate is rate limiting.