Acetoxy drug: protein transacetylase of buffalo liver-characterization and mass spectrometry of the acetylated protein product

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K(m) (1667 microM) and V(max) (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys(51), Lys(82), Lys(123), Lsy(181), Lys(191) and Lys(210)) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.     

Acetoxy drug: protein transacetylase of buffalo liver—characterization and mass spectrometry of the acetylated protein product

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3–3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K_m (1667 μM) and V_max (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3–3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3–3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3–3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3–3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys₅₁, Lys₈₂, Lys₁₂₃, Lsy₁₈₁, Lys₁₉₁ and Lys₂₁₀) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.     

Moonlighting protein in Starkeyomyces koorchalomoides: Characterization of dihydrolipoamide dehydrogenase as a protein acetyltransferase utilizing acetoxycoumarin as the acetyl group donor

In this report we have identified for the first time a transacetylase (TAase) in a mesophilic fungi Starkeyomyces koorchalomoides catalyzing the transfer of acetyl group from polyphenolic acetate (PA) to a receptor protein glutathione S-transferase (GST). An elegant assay procedure was established for TAase based on its ability to mediate inhibition of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model PA. Utilizing this assay procedure, S. koorchalomoides TAase was purified to homogeneity. TAase was found to have MW of 50 kDa. The purified enzyme exhibited maximum activity at 45 °C at pH 6.8. The N-terminal sequence of purified fungal TAase (ANDASTVED) showed identity with corresponding N-terminal sequence of dihydrolipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme and an E3 component of pyruvate dehydrogenase complex (PDHC). TAase was found to have all the properties of LADH and avidly interacted with the anti-LADH antibody. TAase catalyzed acetylation of GST by DAMC was identified by LC–MS/MS and a single lysine residue (Lys-113) was found to be acetylated. Further, recombinant LADH from Streptococcus pneumoniae lacking lipoyl domain was found to exhibit little TAase activity, suggesting the role of lipoyl domain in the TAase activity of LADH. These observations bear evidence for the protein acetyltransferase activity of LADH. Such an activity of LADH can be attributed as a moonlighting function of the enzyme.     

Characterization of protein transacetylase from human placenta as a signaling molecule calreticulin using polyphenolic peracetates as the acetyl group donors

We have earlier shown that a unique membrane-bound enzyme mediates the transfer of acetyl group(s) from polyphenolic peracetates (PA) to functional proteins, which was termed acetoxy drug: protein transacetylase (TAase) because it acted upon several classes of PA. Here, we report the purification of TAase from human placental microsomes to homogeneity with molecular mass of 60 kDa, exhibiting varying degrees of specificity to several classes of PA confirming the structure-activity relationship for the microsome-bound TAase. The TAase catalyzed protein acetylation by a model acetoxy drug, 7,8-diacetoxy-4-methyl coumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with anti-acetyl lysine antibody. TAase activity was severely inhibited in calcium-aggregated microsomes as well as when Ca2+ was added to purified TAase, suggesting that TAase could be a calcium binding protein. Furthermore, the N-terminal sequence analysis of purified TAase (EPAVYFKEQFLD) using Swiss Prot Database perfectly matched with calreticulin (CRT), a major microsomal calcium binding protein of the endoplasmic reticulum (ER). The identity of TAase with CRT was substantiated by the observation that the purified TAase avidly reacted with commercially available antibody raised against the C-terminus of human CRT (13 residues peptide, DEEDATGQAKDEL). Purified TAase also showed Ca2+ binding and acted as a substrate for phosphorylation catalyzed by protein kinase C (PKC), which are hallmark characteristics of CRT. Further, purified placental CRT as well as the commercially procured pure CRT yielded significant TAase catalytic activity and were also found effective in mediating the acetylation of the target protein NADPH cytochrome P-450 reductase by DAMC as detected by Western blot using anti-acetyl lysine antibody. These observations for the first time convincingly attribute the transacetylase function to CRT. Hence, this transacetylase function of CRT is designated calreticulin transacetylase (CRTAase). We envisage that CRTAase plays an important role in protein modification by way of acetylation independent of Acetyl CoA.     

Characterization of protein acyltransferase function of recombinant purified GlnA1 from Mycobacterium tuberculosis: a moon lighting property

The protein acetyltransferase (MTAase) function of glutamine synthetase of Mycobacterium smegmatis was established earlier. In this paper, studies were undertaken to examine MTAase function of recombinant glutamine synthetase (rGlnA1) of Mycobacterium tuberculosis, which showed >80% similarity with M. smegmatis GlnA. The specificity of MTAase to several acyl derivative of coumarins was examined. The results clearly indicated that MTAase exhibited differential specificities to several acyloxycoumarins. Further, MTAase was also found capable of transferring propionyl and butyryl groups from propoxy and butoxy derivatives of 4-methylcoumarin. These observations characterized MTAase in general as a protein acyltransferase. MTAase catalyzed acetylation of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model acetoxy coumarin was confirmed by MALDI-TOF-MS as well as western blot analysis using acetylated lysine polyclonal antibody. In order to validate the active site of rGlnA1 for TAase activity, effect of DAMC and L-methionine-S-sulfoximine (MSO) on GS and TAase activity of rGlnA1 were studied. The results indicated that the active sites of GS and TAase were found different. Acetyl CoA, a universal biological acetyl group donor, was also found to be a substrate for MTAase. These results appropriately characterize glutamine synthetase of Mtb exhibiting transacylase action as a moonlighting protein.     

Establishment of glutamine synthetase of Mycobacterium smegmatis as a protein acetyltransferase utilizing polyphenolic acetates as the acetyl group donors

Acetoxy Drug: Protein Transacetylase (TAase) mediating the transfer of acetyl group(s) from polyphenolic acetates (PA) to certain functional proteins in mammalian cells was identified by our earlier investigations. TAase activity was characterized in the cell lysates of Mycobacterium smegmatis and the purified protein was found to have M(r) 58,000. TAase catalysed protein acetylation by a model acetoxy drug 7,8-diacetoxy-4-methylcoumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with an anti-acetyllysine antibody. The specificity of the TAase of M. smegmatis (MTAase) to various acetoxycoumarins was found to be in the order DAMC > 7-AMC > 6-AMC > 4-AC > 3-AC > ABP. Also, the N-terminal sequence of purified MTAase was found to perfectly match with glutamine synthetase (GS) of M. smegmatis. The identity of MTAase with GS was confirmed by the observation that the purified MTAase as well as the purified recombinant GS exhibited all the properties of GS. The finding that purified Escherichia coli GS was found to have substantial TAase activity highlighted the TAase function of GS in other bacteria. These results conclusively established for the first time the protein acetyltransferase function of GS of M. smegmatis.     

Calreticulin transacylase: Genesis,mechanism of action and biological applications

Our earlier investigations have identified a unique enzyme in the endoplasmic reticulum (ER) termed Acetoxy Drug: Protein Transacetylase (TAase) catalyzing the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP). An elegant assay procedure for TAase was developed based on the inhibition of glutathione S-transferase (GST) due to acetylation by a model acetoxycoumarin, 7, 8-Diacetoxy-4-methylcoumarin (DAMC). TAase purified from various mammalian tissue microsomes to homogeneity exhibited a molecular weight (M.wt) of 55 kDa. Further, by N-terminal sequencing TAase was identified as Calreticulin (CR), a multifunctional Ca²⁺-binding protein in ER lumen. The identity of TAase with CR was evidenced by proteomics studies such as immunoreactivity with anti-CR antibody and mass spectrometry. This function of CR was termed Calreticulin transacetylase (CRTAase). CRTAase was also found to mediate the transfer of acetyl group from DAMC to RP such as NADPH Cytochrome c Reductase (CYPR) and Nitric Oxide Synthase (NOS). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of RP by DAMC was observed. CRTAase activity was found to be inhibited by Ca²⁺. Our investigations on the individual domains (N, P and C) of CR from a nematode Haemonchus contortus revealed that the P-domain alone was found to possess CRTAase activity. Based on the observation that the autoacetylated CR was a stable intermediate in the CRTAase catalyzed protein acetylation by PA, a putative mechanism was proposed. Further, CRTAase was also found capable of transferring propionyl group from a propoxy derivative of polyphenol, 7,8-Dipropoxy-4-methylcoumarin (DPMC) to RP and concomitant autopropionylation of CR was encountered. Hence, CRTAase was assigned the general term Calreticulin Transacylase. Also, CRTAase was found to act upon the biological acyl group donors, acetyl CoA and propionyl CoA. CRTAase mediated modulation of specific functional proteins by way of acylation was exploited to elicit the biological applications of PA.     

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 7: Assay and characterization of 7,8-diacetoxy-4-methylcoumarin:protein transacetylase from rat liver microsomes based on the irreversible inhibition of cytosolic glutathione S-transferase

The enzymatic transfer of acetyl groups from acetylated xenobiotics to specific proteins is a relatively grey area in the evergreen field of biotransformation of foreign compounds. In this paper, we have documented evidence for the existence of a transacetylase in liver microsomes that catalyses the transfer of acetyl groups from 7,8-diacetoxy-4-methylcoumarin (DAMC) to glutathione S-transferase (GST), either purified or present in cytosol leading to the irreversible inhibition of GST. A simple procedure is described for the assay of transacetylase by preincubation of DAMC with liver microsomes and pure GST/liver cytosol, followed by the addition of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GSH) in order to quantify GST activity by the conventional procedure. The extent of inhibition of GST by DAMC under the conditions of the assay is indicative of DAMC:protein transacetylase activity. Following the assay procedure described here, the transacetylase was shown to exhibit hyperbolic kinetics. The bimolecular nature of the transacetylase reaction was apparent by the demonstration of Km and vmax values. 7,8-Dihydroxy-4-methylcoumarin (DHMC), one of the products of transacetylase reaction was identified and quantified using the partially purified enzyme. The fact that p-hydroxymercuribenzoate (PHMB) and iodoacetamide abolished irreversible inhibition of GST upon the action of transacetylase on DAMC strongly characterized transacetylase as a protein containing thiol group at the active site. In addition, the relative specificities of acetoxy 4-methylcoumarins to transacetylase have been demonstrated.     

Mechanism of biochemical action of substituted 4-methylcoumarins. Part 11: Comparison of the specificities of acetoxy derivatives of 4-methylcoumarin and 4-phenylcoumarin to acetoxycoumarins: protein transacetylase

Our earlier observations led to the identification of a microsomal enzyme termed as acetoxy drug: protein transacetylase (TAase) catalyzing the transfer of acetyl groups from acetylated polyphenols to the receptor proteins. TAase was conveniently assayed by the irreversible inhibition of cytosolic glutathione S-transferase (GST) by the model acetoxycoumarin, 7,8-diacetoxy-4-methylcoumarin (1). The specificities of the acetoxy group on the benzenoid ring and position of the pyran carbonyl group of the coumarin with respect to oxygen heteroatom for the catalytic activity of TAase were also reported earlier. In this communication, we have demonstrated that the acetoxy coumarins and acetoxy dihydrocoumarins having a methyl group instead of a phenyl ring at the C-4, when used as the substrates, resulted in enhancement of TAase activity, while the saturation of double bond at C-3 and C-4 position had no effect on TAase activity. A comparison of the optimized structures of 1 and 7,8-diacetoxy-4-phenylcoumarin (2) suggested that the observed influence may be due to out of plane configuration of the phenyl ring at C-4. Further, the TAase-catalyzed activation of NADPH cytochrome c reductase and inhibition of aflatoxin B1 (AFB1)-DNA binding by acetoxy 4-phenylcoumarins and dihydrocoumarins were significantly lower as compared to those caused by acetoxy 4-methylcoumarins.     

Nonenzymatic acetylation of histones with acetyl phosphate and acetyl adenylate.

Nonenzymatic acetylation of calf-thymus lysine- and arginine-rich histones was demonstrated to occur when these proteins were incubated with [14C]acetyl phosphate and [14C]acetyl adenylate. The levels of acetylation depend on both pH and on reagent concentration. When acetyl [33P]phosphate and acetyl [3H]adenylate were used as reagents, we found neither histone phosphorylation nor adenylylation. Most of the radioactivity of 14C-labeled acetylated histones was recovered as Ne-acetyllysine. Furthermore, only a small amount of O-bound radioactivity was released by the 14C-labeled acetylated arginine-rich histone during treatment with hydroxylamine. Experiments on the acetylation of histones, in the presence of increasing salt concentration, gave different results for the two acetylating agents.     

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 10: identification of inhibitors for the liver microsomal acetoxycoumarin: protein transacetylase

The quantitative structure-activity relationship (QSAR) studies conducted by us earlier revealed the cardinal role of the pyran ring carbonyl group in the acetoxy polyphenolic compounds for the acetoxy polyphenol:protein transacetylase (TAase) activity. Hence, an attempt was made to examine whether such substrate analogues of benzopyran acetates which lack in the pyran ring carbonyl group, such as 7-acetoxy-2,3-dihydro-2,2-dimethylbenzopyran (BPA), cetachin pentaacetate (CPA) and hematoxylin pentaacetate (HPA) could inhibit the 7,8-diacetoxy-4-methylcoumarin (DAMC):protein (glutathione-S-transferase) transacetylase activity. These compounds were indeed found to remarkably inhibit the TAase activity in a concentration dependent manner and exerted their inhibitory action very rapidly. Further BPA, CPA and HPA were found to abolish the TAase mediated activation of NADPH cytochrome C reductase as well as the inhibition of liver microsome catalyzed aflatoxin B(1) (AFB(1))-DNA binding by DAMC very effectively. These results strongly suggest that the acetoxybenzopyrans merit as potent inhibitors of TAase.     

The yeast fatty acid synthase. Pathway for transfer of the acetyl group from coenzyme A to the Cys-SH of the condensation site

The reaction pathway of enzyme-catalyzed acetylation of the acyl-accepting sites of the yeast synthase, a Ser-OH at the acetyl transacylase site, a Cys-SH at the beta-ketoacyl synthase site, and the acyl carrier protein 4'-phosphopantetheine-SH (Pant-SH), has been investigated using the chromophoric substrate, p-nitrophenyl thioacetate. The stoichiometry of acetylation of the native enzyme was 3 mol of acetate bound per mol of synthase unit, alpha beta (Mr 430,000). The acetylation process is biexponential; the rate constant of acetylation of the first 2 mol is 5.0 s-1 and the third mol is 0.2 s-1. The pathway by which acetyl moiety is added to the enzyme was determined by selectively blocking the acyl-accepting sites and subsequently determining the kinetics and stoichiometry of acetylation. The dibromopropanone-treated enzyme, in which the Pant-SH and Cys-SH are alkylated, exhibited an exponential burst of approximately 1 mol/mol of synthase unit with a rate constant of 11.0 s-1. The iodoacetamide-treated enzyme, in which Cys-SH is alkylated, had a biexponential burst with a total stoichiometry of approximately 2 mol/mol of synthase unit, with rate constants of 9 and 0.2 s-1, respectively. The kinetically competent acetylation to the extent of 2 and approximately 1 mol/mol of synthase unit for both Cys-SH and Cys-SH and Pant-SH-blocked enzymes, respectively, indicated that the route of acetyl transfer in the yeast synthase is obligatorily Ser-OH----Cys-SH. The acetylation of Pant-SH (0.2 s-1) occurs with a rate insignificant to the process of fatty acid synthesis (turnover rate constant of 1.5 s-1). These conclusions are supported by experiments involving end point radiolabeling of the synthase with [1-14C]acetyl moieties using the substrate, p-nitrophenyl thio[1-14C]acetate. Native, dibromopropanone-treated, and iodoacetamide-treated enzymes bind about 3, 1, and 2 mol of acetyl/mol of synthase unit, respectively. Performic acid oxidation studies of the acetyl-labeled enzyme indicate that there is one Ser-O-acetyl formed in the native and alkylated enzymes and one Cys-S-acetyl and one Pant-S-acetyl formed in the native enzyme. Altogether, these results support our contention that the acetylation of the Pant-SH is kinetically incompetent. Thus, the yeast synthase transacetylation reactions occur by a novel process of acetyl transfer from CoA to Ser-OH----Cys-SH, which is in contrast to the transfer from CoA to Ser-OH----Pant-SH----Cys-SH catalyzed by the prokaryotic synthases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reaction of the anionic acetylation agent methyl acetyl phosphate with D-3-hydroxybutyrate dehydrogenase

Methyl acetyl phosphate is a competitive inhibitor of the reduction of acetoacetate by D-3-hydroxybutyrate dehydrogenase. The material also irreversibly inactivates the enzyme. The kinetics of the inactivation are consistent with methyl acetyl phosphate acetylating the conjugate base of a hydrogen bond donor. Protection offered by a substrate analogue (methyl acetonylphosphonate) in the presence of coenzyme implicates reaction at the cationic active site. Reversible protection by the amino group reagent 2,3-dimethylmaleic anhydride suggests that methyl acetyl phosphate reacts with an amino group. Sulfhydryl reagents and acetyl phosphate, a poorer acetylating agent, do not inactivate the enzyme. The pH dependence of the inactivation suggests that the acetylation occurs at a site that has a pKa of 8.2. The utility of methyl acetyl phosphate and other acyl phosphate monoesters in reacting with lysines adjacent to cationic sites of enzymes, hemoglobin, and histones is noted.     

An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A

1. Purified 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast (free from acetoacetyl-CoA thiolase activity) catalysed an exchange of acetyl moiety between 3'-dephospho-CoA and CoA. The exchange rate was comparable with the overall velocity of synthesis of 3-hydroxy-3-methylglutaryl-CoA. 2. Acetyl-CoA reacted with the synthase, giving a rapid ;burst' release of CoA proportional in amount to the quantity of enzyme present. The ;burst' of CoA was released from acetyl-CoA, propionyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA) but not from acetoacetyl-CoA, hexanoyl-CoA, dl-3-hydroxy-3-methylglutaryl-CoA, or other derivatives of glutaryl-CoA. 3. Incubation of 3-hydroxy-3-methylglutaryl-CoA synthase with [1-(14)C]acetyl-CoA yielded protein-bound acetyl groups. The K(eq.) for the acetylation was 1.2 at pH7.0 and 4 degrees C. Acetyl-labelled synthase was isolated free from [1-(14)C]acetyl-CoA by rapid gel filtration at pH6.1. The [1-(14)C]acetyl group was removed from the protein by treatment with hydroxylamine, CoA or acetoacetyl-CoA but not by acid. When CoA or acetoacetyl-CoA was present the radioactive product was [1-(14)C]acetyl-CoA or 3-hydroxy-3-methyl-[(14)C]glutaryl-CoA respectively. 4. The isolated [1-(14)C]acetyl-enzyme was slowly hydrolysed at pH6.1 and 4 degrees C with a first-order rate constant of 0.005min(-1). This rate could be stimulated either by raising the pH to 7.0 or by the addition of desulpho-CoA. 5. These properties are interpreted in terms of a mechanism in which 3-hydroxy-3-methyl-glutaryl-CoA synthase is acetylated by acetyl-CoA to give a stable acetyl-enzyme, which then condenses with acetoacetyl-CoA yielding a covalent derivative between 3-hydroxy-3-methylglutaryl-CoA and the enzyme which is then rapidly hydrolysed to free enzyme and product.     

3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Properties of its acetyl derivative.

Ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) reacts with acetyl-CoA to form a complex in which the acetyl group is covalently bound to the enzyme. This acetyl group can be removed by addition of acetoacetyl-CoA or CoA. The extent of acetylation and release of CoA were found to be highly temperature-dependent. At temperatures above 20 degrees C, a maximum value of 0.85 mol of acetyl group bound/mol of enzyme dimer was observed. Below this temperature the extent of rapid acetylation was significantly lowered. Binding stoichiometries close to 1 mol/mol of enzyme dimer were also observed when the 3-hydroxy-3-methylglutaryl-CoA synthase activity was titrated with methyl methanethiosulphonate or bromoacetyl-CoA. This is taken as evidence for a 'half-of-the-sites' reaction mechanism for the formation of 3-hydroxy-3-methylglutaryl-CoA by 3-hydroxy-3-methylglutaryl-CoA synthase. The Keq. for the acetylation was about 10. Isolated acetyl-enzyme is stable for many hours at 0 degrees C and pH 7, but is hydrolysed at 30 degrees C with a half-life of 7 min. This hydrolysis is stimulated by acetyl-CoA and slightly by succinyl-CoA, but not by desulpho-CoA. The site of acetylation has been identified as the thiol group of a reactive cysteine residue by affinity-labelling with the substrate analogue bromo[1-14C]acetyl-CoA.     

3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.     

Acetoxy drug: protein transacetylase catalyzed activation of human platelet nitric oxide synthase by polyphenolic peracetates

An enhanced intracellular level of Nitric oxide (NO) is essential to ameliorate several pathological conditions of heart and vasculature necessitating the activation of NOS. We have projected in this report the acetylation of eNOS by polyphenolic peracetates (PA) catalyzed by the novel enzyme acetoxy drug: protein transacetylase (TAase) discovered in our laboratory as an unambiguous way of activating NOS which results in the manifestation of physiological action. The human platelet was chosen as the experimental system in order to validate the aforementioned proposition. PA caused profound irreversible activation of platelet NADPH cytochrome c reductase mediated by TAase. The convincing biochemical evidences are presented to show that PA could cause acetylation of the reductase domain of NOS leading to the activation of eNOS in tune with their specificities to platelet TAase. As a result, the enhanced level of NO due to activation of platelet eNOS by PA was found to inhibit the ADP-induced platelet aggregation. The present studies highlight for the first time the role of PA as the novel potent agent for enhancing the intracellular NO levels.     

Molecular cloning, and characterization of a modular acetyl xylan esterase from the edible straw mushroom Volvariella volvacea

A new Volvariella volvacea gene encoding an acetyl xylan esterase (designated as Vvaxe1) was cloned and expressed in Pichia pastoris. The cDNA contained an ORF of 1047 bp encoding 349 amino acids with a calculated mass of 39 990 Da. VvAXE1 is a modular enzyme consisting of an N-terminal signal peptide, a catalytic domain, and a cellulose-binding domain. The amino acid sequence of the enzyme exhibited a high degree of similarity to cinnamoyl esterase B from Penicillium funiculosum, and acetyl xylan esterases from Aspergillus oryzae, Penicillium purpurogenum, and Aspergillus ficuum. Recombinant acetyl xylan esterase released acetate from several acetylated substrates including beta-d-xylose tetraacetate and acetylated xylan. No activity was detectable on p-nitrophenyl acetate. Enzyme-catalyzed hydrolysis of 4-methylumbelliferyl acetate was maximal at pH 8.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 307.7 microM and a V(max) value of 24 733 IU micromol(-1) protein. ReAXE1 also exhibited a capacity to bind to Avicel and H(3)PO(4) acid-swollen cellulose.     

The TRIM family protein KAP1 inhibits HIV-1 integration

The integration of viral cDNA into the host genome is a critical step in the life cycle of HIV-1. This step is catalyzed by integrase (IN), a viral enzyme that is positively regulated by acetylation via the cellular histone acetyl transferase (HAT) p300. To investigate the relevance of IN acetylation, we searched for cellular proteins that selectively bind acetylated IN and identified KAP1, a protein belonging to the TRIM family of antiviral proteins. KAP1 binds acetylated IN and induces its deacetylation through the formation of a protein complex which includes the deacetylase HDAC1. Modulation of intracellular KAP1 levels in different cell types including T cells, the primary HIV-1 target, revealed that KAP1 curtails viral infectivity by selectively affecting HIV-1 integration. This study identifies KAP1 as a cellular factor restricting HIV-1 infection and underscores the relevance of IN acetylation as a crucial step in the viral infectious cycle.     

Acetylation of Amino Groups and Its Effect on the Structure of Soybean Glycinin

Purified soybean glycinin (11S globulin) was acetylated at three degrees; low (21-51%), middle (60-81%) and high (90-92%) acetylation in lysine residues. With increasing the acetyl content, the β-structure gradually decreased and the random structure increased resulting in the exposure of tyrosine residue. These were determined from the results of optical rotatory dispersion, intrinsic viscosity, ultraviolet and fluorescence measurement. Gel filtration, ultracentrifugation and gel electrophoresis studies showed drastic conformational changes of highly acetylated 11S (over 90%), in which most of the modified protein (75%) polymerized, and the other dissociated into 3S protein. The close relation between the conformation of acetylated 11S and its emulsifying properties was discussed.