Human plasma lecithin-cholesterol acyltransferase. An elucidation of the catalytic mechanism

Human plasma lecithin-cholesterol acyltransferase (LCAT) transacylates the sn-2 fatty acid of lecithin to cholesterol forming cholesteryl ester and lysolecithin. Measurement of the phospholipase A2 and transacylase activities of the enzyme using proteoliposome substrates and following selective chemical modification of serine, histidine, and cysteine residues of pure homogeneous LCAT indicated the following catalytic mechanism: HS-Cys-E-Ser-OH + lecithin in equilibrium HS-Cys-E-Ser-O-FA + lysolecithin, HS-Cys-E-Ser-O-FA in equilibrium FA-S-Cys-E-Ser-OH, FA-S-Cys-E-Ser-OH + cholesterol-OH in equilibrium HS-Cys-E-Ser-OH + cholesterol-O-FA, where FA denotes fatty acid. Modification of 2 LCAT cysteine residues with 5,5'-dithiobis-(2-nitrobenzoic acid) or treatment with ferricyanide inactivated the transacylase but not the phospholipase A2 activity. Modification of 1 serine residue with phenylmethanesulfonyl fluoride or 1 histidine residue with diethyl pyrocarbonate inhibited cholesteryl ester formation and phospholipase A2 activity. Proteoliposome substrates protected both activities against chemical inactivation. Lecithin alone protected the phospholipase A2 activity against phenylmethanesulfonyl fluoride inactivation but not the transacylase against 5,5'-dithiobis-(2-nitrobenzoic acid) inactivation. Incubation of native LCAT with arachidonyl-CoA or the lecithin-apo-A-I proteoliposome resulted in acylation of three enzyme sites, only one of which was stable to neutral hydroxylamine after denaturation. Fatty acylenzyme oxy- and thioesters were demonstrable in both cases. No transfer of arachidonic acid from iodoacetamide-modified LCAT to cholesterol occurred, indicating that the fatty-acylated serine residue cannot directly esterify cholesterol. Cholesterol arachidonate was formed upon incubation of phenylmethanesulfonyl fluoride-modified LCAT with arachidonyl-CoA.     

Affinity labeling of cofactor-binding region of human placental estradiol 17 beta-dehydrogenase by periodate-oxidized NADP+ (o-NADP+)

Periodate-oxidized NADP+ (o-NADP+), an analogue of the cofactors, is a reversible inhibitor of estradiol 17 beta-dehydrogenase in human placenta. Mode of the inhibition by o-NADP+ appeared to be competitive type (Ki = 0.84 microM) against NAD+ and non-competitive type (Ki = 1.13 microM) against estradiol, respectively. Treatment of the estradiol 17 beta-dehydrogenase with o-NADP+ resulted in time-dependent loss of the enzyme activity. The inactivation exhibited pseudo-first order kinetics (t1/2 = 15 min) and was protected by NAD+ and NADP+. On the other hand, periodate-oxidized ATP inactivated slightly the estradiol 17 beta-dehydrogenase. These results indicate that the residue(s) of lysines is located near the cofactor-binding region of estradiol 17 beta-dehydrogenase of human placenta.     

Evidence that lipid hydroperoxides inhibit plasma lecithin:cholesterol acyltransferase activity

The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. Recently, we found that polar lipids isolated from minimally oxidized LDL produced a dramatic inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, suggesting that HDL-cholesterol transport may be impaired during early atherogenesis. In this study, we have identified molecular species of oxidized lipids that are potent inhibitors of LCAT activity. Treatment of LDL with soybean lipoxygenase generated small quantities of lipid hydroperoxides (20 +/- 4 nmol/mg LDL protein, n = 3); but when lipoxygenase-treated LDL (1 mg protein/ml) was recombined with the d > 1.063 g/ml fraction of human plasma, LCAT activity was rapidly inhibited (25 +/- 4 and 65 +/- 16% reductions by 1 and 3 h, respectively). As phospholipid hydroperoxides (PL-OOH) are the principal oxidation products associated with lipoxygenase-treated LDL, we directly tested whether PL-OOH inhibited plasma LCAT activity. Detailed dose-response curves revealed that as little as 0.2 and 1.0 mole % enrichment of plasma with PL-OOH produced 20 and 50% reductions in LCAT activity by 2 h, respectively. To gain insight into the mechanism of LCAT impairment, the enzyme's free cysteines (Cys31 and Cys184) and active site residues were "capped" with the reversible sulfhydryl compound, DTNB, during exposure to either minimally oxidized LDL or PL-OOH. Reversal of the DTNB "cap" after such exposures revealed that the enzyme was completely protected from both sources of peroxidized phospholipids. We, therefore, conclude that PL-OOH inhibited plasma LCAT activity by modifying the enzyme's free cysteine and/or catalytic residues. These studies are the first to suggest that PL-OOH may accelerate the atherogenic process by impairing LCAT activity.     

Inactivation of Trypanosoma cruzi and Crithidia fasciculata topoisomerase I by Fenton systems

Fenton systems (H(2)O(2)/Fe(II) or H(2)O(2)/Cu(II)) inhibited Trypanosoma cruzi and Crithidia fasciculata topoisomerase I activity. About 61-71% inactivation was produced by 25 microM Fe(II) or Cu(II) with 3.0 mM H(2)O(2). Thiol compounds and free radical scavengers prevented Fenton system effects, depending on the topoisomerase assayed. With the T. cruzi enzyme, reduced glutathione (GSH), dithiothreitol (DTT), cysteine and N-acetyl-L-cysteine (NAC) entirely prevented the effect of the H(2)O(2)/Fe(II) system; mannitol protected 37%, whereas histidine and ethanol were ineffective. With C. fasciculata topoisomerase, GSH, DTT and NAC protected 100%, cysteine, histidine and mannitol protected 28%, 34% and 48%, respectively, whereas ethanol was ineffective. With the H(2)O(2)/Cu(II) system and T. cruzi topoisomerase, DTT and histidine protected 100% and 60%, respectively, but the other assayed protectors were less effective. Similar results were obtained with the C. fasciculata enzyme. Topoisomerase inactivation by the H(2)O(2)/Fe(II) or H(2)O(2)/Cu(II) systems proved to be irreversible since it was not reversed by the more effective enzyme protectors. It is suggested that topoisomerases could act either as targets of 'reactive oxygen species' (ROS) generated by Fenton systems or bind the corresponding metal ions, whose redox cycling would generate reactive oxygen species in situ.     

Enzyme IIMtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier

The cysteines of the membrane-bound mannitol-specific enzyme II (EIIMtl) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity [Roossien, F. F., & Robillard, G. T. (1984) Biochemistry 23, 211-215]. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with [14C]iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing.     

NAD(H) enhances the Cu(II)-mediated inactivation of lactate dehydrogenase by increasing the accessibility of sulfhydryl groups

Copper ions are known to inactivate a variety of enzymes, and lactate dehydrogenase (LDH) is exceptionally sensitive to the presence of this metal. We now found that NADH strongly enhances the Cu(II)-mediated loss of LDH activity. Surprisingly, NADH was not oxidized in this process and also NAD⁺ promoted the Cu(II)-dependent inactivation of LDH. Catalase only partly protected the enzyme, whereas hypoxia even enhanced LDH inactivation. NAD(H) accelerated sulfhydryl (SH) group oxidation of LDH by 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), and, vice versa, LDH-mediated Cu(II) reduction. LDH activity was preserved by thiol donators and pyruvate and partially preserved by lactate and oxamate. Our results suggest that reactive oxygen species (ROS) are of minor importance for the inactivation of LDH induced by Cu(II)/NADH. We propose that conformational changes of the enzymes' active sites induced by NAD(H)-binding increase the accessibility of active sites' cysteine residues to Cu(II) thereby accelerating their oxidation and, consequently, loss of catalytic activity.     

Effects of di-isopropyl phosphorofluoridate on rat liver microsomal and lysosomal beta-glucuronidase

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.     

Redox modulation of Rubisco conformation and activity through its cysteine residues

Treatment of purified Rubisco with agents that specifically oxidize cysteine-thiol groups causes catalytic inactivation and increased proteolytic sensitivity of the enzyme. It has been suggested that these redox properties may sustain a mechanism of regulating Rubisco activity and turnover during senescence or stress. Current research efforts are addressing the structural basis of the redox modulation of Rubisco and the identification of critical cysteines. Redox shifts result in Rubisco conformational changes as revealed by the alteration of its proteolytic fragmentation pattern upon oxidation. In particular, the augmented susceptibility of Rubisco to proteases is due to increased exposure of a small loop (between Ser61 and Thr68) when oxidized. Progressive oxidation of Rubisco cysteines using disulphide/thiol mixtures at different ratios have shown that inactivation occurs under milder oxidative conditions than proteolytic sensitization, suggesting the involvement of different critical cysteines. Site-directed mutagenesis of conserved cysteines in the Chlamydomonas reinhardtii Rubisco identified Cys449 and Cys459 among those involved in oxidative inactivation, and Cys172 and Cys192 as the specific target for arsenite. The physiological importance of Rubisco redox regulation is supported by the in vivo response of the cysteine mutants to stress conditions. Substitution of Cys172 caused a pronounced delay in stress-induced Rubisco degradation, while the replacement of the functionally redundant Cys449-Cys459 pair resulted in an enhanced catabolism with a faster high-molecular weight polymerization and translocation to membranes. These results suggest that several cysteines contribute to a sequence of conformational changes that trigger the different stages of Rubisco catabolism under increasing oxidative conditions.     

Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis decays steadily when exposed to agents that induce oxidative modification of cysteine residues (Cu²⁺, benzofuroxan, disulfides, arsenite, oxidized ascorbate). Inactivation takes place with a concomitant loss of cysteine sulfhydryl groups and dimerization of large subunits of the enzyme. 40% activity loss induced by the vicinal thiol-reagent arsenite is caused by modification of a few neighbor residues while the almost complete inactivation achieved with disulfides is due to extensive oxidation leading to formation of mixed disulfides with critical cysteines of the protein. In most cases oxidative inactivation is also accompanied by an increased sensitivity to proteolysis by trypsin, chymotrypsin or proteinase K. Both enzymatic activity and resistance to proteolysis can be restored through treatment with several thiols (cysteamine, cysteine, dithiothreitol and, more slowly, reduced glutathione). Redox effectors which are thought to regulate the chloroplast activity (NADPH, ferredoxin and thioredoxin) do not reactivate the oxidized enzyme. When ribulose-1,5-bisphoshate carboxylase/oxygenase is incubated with cystamine/cysteamine mixtures having different disulfide/thiol ratio (r), inactivation takes place around r=1.5 while proteolytic sensitization occurs under more oxidative conditions (r=4). It is suggested that oxidative modification may happen in vivo under exceptional circumstances, such as senescence, bleaching or different kinds of stress, leading to enzyme inactivation and triggering the selective degradation of the carboxylase that has been repeatedly observed during these processes.     

Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein from Escherichia coli: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes

The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme from Escherichia coli was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. The K_m values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.     

Studies on the reactivity of the essential sulfhydryl groups as a conformational probe for the fatty acid synthetase of chicken liver. Inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid) and intersubunit cross-linking of the inactivated enzyme

Fatty acid synthetase of chicken liver is rapidly and reversibly inactivated by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) at a rate (k2 = 132 mM-1 S-1 in 3 mM EDTA, 1% (v/v) glycerol, pH 7.0, at 25 degrees C) up to 2200 times higher than the reaction of this reagent with simple thiol compounds. The inactivation is caused by the reaction of the phosphopantetheine SH group, since it is protected competitively by either acetyl- or malonyl-CoA, and since the inactivated enzyme is unreactive with the phosphopantetheine label chloroacetyl-CoA but reactive with the cysteine reagent 1,3-dibromopropanone. Moreover, chloroacetyl-CoA prevents the modification of the rapidly reacting essential SH group by DTNB. The number of SH groups involved in inactivation was determined by correlating activity loss with the extent of reaction and by stopped-flow analysis of substrate (or chloroacetyl-CoA) protection. Values between 0.91 and 1.15 SH groups/dimer were obtained, indicating the presence of substoichiometric amounts of the prosthetic group in the fatty acid synthetase preparations used in this study. Inactivation of the synthetase by DTNB is strongly inhibited by increasing salt concentration and protected noncompetitively by NADP+ and NADPH. Treatment of the enzyme inactivated at low salt by salt, NADP+, or NADPH also effectively reduced cross-linking between enzyme subunits. The parallel effects of these treatments on the reaction with DTNB and subsequent dimerization are consistent with a minimum model of two discreet conformation states for fatty acid synthetase. In the low salt conformer, the phosphopantetheine and cysteine SH groups are juxtaposed, and the DTNB reaction (k2 approximately 132 mM-1 S-1) and dimerization are both facilitated. Transition to the high salt conformer by the above treatments is accompanied by an approximately 20-fold reduction of reactivity with DTNB (k2 = 6.8 mM-1 S-1) and reduced dimerization, due to spatial separation of the SH groups. During palmitate synthesis, the enzyme may oscillate between these conformation states to permit the reaction of intermediates at different active sites. Results obtained by studies on the effect of pH on DTNB inactivation implicate a pK of 5.9-6.1 for the essential SH group independent of salt concentration. This value is 1.5-1.8 pH units lower than the pK of 7.6-7.7 for CoA and may explain the 23-fold increase of the rate constant from a value of 0.3 mM-1 S-1 for CoA to that of the high salt conformer.     

Interaction of glutathione transferase from horse erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with two thiol groups of the dimeric horse erythrocyte glutathione transferase at pH 5.0, with strong inactivation reversible on dithiothreitol treatment. The inactivation kinetic follows a biphasic pattern, similar to that caused by other thiol reagents as recently reported. Both S-methylglutathione and 1-chloro-2,4-dinitrobenzene protect the enzyme from inactivation. Analysis of the reactive SH group-containing peptide gives the sequence Ala-Ser-Cys-Leu-Tyr, identical with that of the peptide that contains the reactive cysteine 47 of the human placental transferase. In the presence of glutathione, the enzyme is not inactivated by this reagent, but it catalyzes its conjugation to glutathione. At higher pH values, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with 2 tyrosines/dimer and lysines, as well as with cysteines. Reaction with lysine seems essentially without effect on activity; whether the reactive tyrosines are important for activity could not be determined using this reagent only. However, 2 tyrosines among the 4 that are nitrated by tetranitro-methane are important for activity.     

Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione

Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with approximately 3 microM ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S]2+ form to the inactive [3Fe-4S]1+ form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfonic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.     

Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward alpha- and beta-substrate lipoproteins

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the Delta 1 and Delta 2 mutants retain their ability to bind substrate, other factor(s), such as decreased access to the substrate binding pocket, may be responsible for the loss of enzyme activity.     

Inactivation of Buckwheat α-Glucosidase with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

The amino acid residue(s) involved in the activity of buckwheat α-glucosidase was modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester. The modification resulted in the decrease in the hydrolytic activity of the enzyme following pseudo-first order kinetics. Competitive inhibitors, such as Tris and turanose, protected the enzyme against the inactivation. Protection was provided also by alkali metal, alkaline-earth metal and ammonium ions, though these cations are non-essential for the activity of the enzyme. Turanose or K⁺ protected one carboxyl group per enzyme from the modification with carbodiimide and glycine ethyl ester. Free sulfhydryl group of the enzyme was also partially modified with carbodiimide, but the inactivation was considered to be mainly attributed to the modification of essential carboxyl group rather than to that of free sulfhydryl group.     

Mutant acyl-coenzyme A:cholesterol acyltransferase 1 devoid of cysteine residues remains catalytically active.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis and in the early stages of atherosclerosis. ACAT1 is an integral membrane protein with multiple transmembrane domains. Human ACAT1 contains nine cysteine residues; its activity is severely inhibited by various thiol-specific modification reagents including p-chloromercuribenzene sulfonic acid, suggesting that certain cysteine residue(s) might be near or at the active site. We constructed various ACAT1 mutants that contained either single cysteine to alanine substitution at various positions, contained a reduced number of cysteines, or contained no cysteine at all. Each of these mutants retained 20% or more of the wild-type ACAT activity. Therefore, cysteine is not essential for ACAT catalysis. For the cysteine-free enzyme, its basic kinetic properties and intracellular localization in Chinese hamster ovary cells were shown to be very similar to those of the wild-type enzyme. The availability of the cysteine-free ACAT1 will facilitate future ACAT structure function studies. Additional studies show that Cys467 is one of the major target sites that leads to p-chloromercuribenzene sulfonic acid-mediated ACAT1 inactivation, suggesting that Cys467 may be near the ACAT active site(s).     

Ozone-induced inactivation of antioxidant enzymes

Ozone is an air pollutant that damages a variety of biomolecules. We investigated ozone-induced inactivation of three major antioxidant enzymes. Cu/Zn superoxide dismutase was inactivated by ozone in a concentration-dependent manner. The concentration of ozone for 50% inactivation was approximately 45 microM when 10 microM Cu/Zn superoxide dismutase was incubated for 30 min in the presence of ozone. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was randomly fragmented. Both ascorbate and glutathione were very effective in protecting Cu/Zn superoxide dismutase from ozone-induced inactivation. The other two enzymes, catalase and glutathione peroxidase, were much more resistant to ozone than Cu/Zn superoxide dismutase. The ozone concentrations for 50% inactivation of 10 microM catalase and glutathione peroxidase were 500 and 240 microM, respectively. SDS-PAGE demonstrated that ozone caused formation of high molecular weight aggregates in catalase and dimerization in glutathione peroxidase. Glutathione protected catalase and glutathione peroxidase from ozone but the effective concentrations were much higher than that for Cu/Zn superoxide dismutase. Ascorbate was almost ineffective. The result suggests that, among the three antioxidant enzymes, Cu/Zn superoxide dismutase is a major target for ozone-induced inactivation and both glutathione and ascorbate are very effective in protecting the enzyme from ozone.     

Catalytic zinc site and mechanism of the metalloenzyme PR-AMP cyclohydrolase

The enzyme N(1)-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn(2+) metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis, an uncommon ring-opening of a purine heterocycle for use in primary metabolism. A three-dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn(2+) binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate N(1)-(5'-phosphoribosyl)adenosine 5'-monophosphate; (PR-AMP), while Mg(2+), a metal required for catalytic activity, enhanced the rate of inactivation. Site-directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn(2+) content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity, and only the C116A was still capable of binding Zn(2+). Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn(2+) and provides further evidence that the Zn(2+) coordination site involves the three conserved cysteine residues. The C93 reactivity is modulated by the presence of the Zn(2+) and Mg(2+) and substantiates the role of this residue as a metal ligand. In addition, Mg(2+) ligand binding site(s) indicated by the structural analysis were probed by site-directed mutagenesis of three key aspartate residues flanking the conserved C93 which were shown to have a functional impact on catalysis, cysteine activation, and metal (zinc) binding capacity. The unique amino acid sequence, the dynamic properties of the cysteine ligands involved in Zn(2+) coordination, and the requirement for a second metal (Mg(2+)) are discussed in the context of their roles in catalysis. The results are consistent with a Zn(2+)-mediated activation of H(2)O mechanism involving histidine as a general base that has features similar to but distinct from those of previously characterized purine and pyrimidine deaminases.     

Purification and characterization of N-acetylneuraminic acid-9-phosphate synthase from rat liver

Sialic acids are a group of carboxylated amino sugars important for a variety of cellular functions. N-Acetylneuraminic acid (Neu5Ac) is the predominant sialic acid in nature. Neu5Ac-9-phosphate synthase catalyzes the formation of Neu5Ac-9-phosphate from N-acetylmannosamine-6-phosphate and phosphoenolpyruvate. Neu5Ac-9-phosphate synthase was purified 11,700-fold from rat liver cytosol to apparent homogeneity by ammonium sulfate precipitation, chromatography on hydroxylapatite, phenyl-Sepharose, MonoQ, and finally gel filtration. SDS-PAGE and gel filtration chromatography indicated that the enzyme is a dimer composed of 37-kDa subunits. Analysis of trypic peptides by MALDI-TOF MS verified a high sequence similarity to the corresponding murine enzyme. The K(m) values of Neu5Ac-9-phosphate synthase were 35 microM for N-acetylmannosamine-6-phosphate and 100 microM for phosphoenolpyruvate. The enzyme displayed an absolute requirement for divalent cations, Mn(2+), Fe(2+), and Mg(2+) being the most effective. In contrast to human Neu5Ac-9-phosphate synthase, the rat enzyme did not utilize mannose-6-phosphate in the synthesis of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid 9-phosphate. Neu5Ac-9-phosphate synthase was inactivated by the sulfhydryl modifying reagents, 5,5'-dithio-bis (2-nitrobenzoic acid) and N-ethylmaleimide, and protected from inactivation by the presence of the substrate phosphoenolpyruvate, but not by the presence of N-acetylmannosamine-6-phosphate, showing that at least one cysteine residue is located in the active site of the enzyme.     

Specific and reversible inactivation of Phycomyces blakesleeanus isocitrate lyase by ascorbate-iron: role of two redox-active cysteines

Phycomyces blakesleeanus isocitrate lyase (EC 4.1.3.1) is in vivo reversibly inactivated by hydrogen peroxide. The purified enzyme showed reversible inactivation by an ascorbate plus Fe(2+) system under aerobic conditions. Inactivation requires hydrogen peroxide; was prevented by catalase, EDTA, Mg(2+), isocitrate, GSH, DTT, or cysteine; and was reversed by thiols. The ascorbate served as a source of hydrogen peroxide and also reduced the Fe(3+) ions produced in a "site-specific" Fenton reaction. Two redox-active cysteine residues per enzyme subunit are targets of oxidative modification; one of them is located at the catalytic site and the other at the metal regulatory site. The oxidized enzyme showed covalent and conformational changes that led to inactivation, decreased thermal stability, and also increased inactivation by trypsin. These results represent an example of redox regulation of an enzymatic activity, which may play a role as a sensor of redox cellular status.