Chemical modifications of alpha1,6-fucosyltransferase define amino acid residues of catalytic importance

alpha1,6-Fucosyltransferase (alpha6FucT) of human platelets was subjected to the action of phenylglyoxal (PLG), pyridoxal-5'-phosphate/NaBH(4) (PLP), and diethyl pyrocarbonate (DEPC) the reagents that selectively modify the structure of amino acids arginine, lysine and histidine, respectively, as well as to N-ethylmaleimide (NEM), mersalyl, p-chloromercuribenzoate (pCMB), iodoacetate, iodoacetamide, and methyl iodide that react with sulfhydryl group of cysteine. In addition, we treated the enzyme with beta-mercaptoethanol, a reagent that disrupts disulfide bonds. All reagents except NEM significantly inactivated alpha6FucT. Protection against the action of PLG, PLP and sulfhydryl modifying reagents was offered by GDP-fucose, GDP, and the acceptor substrate, a transferrin-derived biantennary glycopeptide with terminal GlcNAc residues. Neither donor nor acceptor substrate offered, however, any protection against inactivation by DEPC or beta-mercaptoethanol. We conclude that arginine, cysteine and probably lysine residues are present in, or closely by, the donor and acceptor substrate binding domains of the enzyme, whereas histidine may be a part of its catalytic domain. However, the primary structure of alpha6FucT does not show cysteine residues in proximity to the postulated GDP-fucose-binding site and acceptor substrate binding site of the enzyme that contains two neighboring arginine residues and one lysine residue (Glycobiol. 10 (2000) 503). To rationalize our results we postulate that platelet alpha6FucT is folded through disulfide bonds that bring together donor/acceptor-binding- and cysteine- and lysine-rich, presumably acceptor substrate binding sites, thus creating a catalytic center of the enzyme.     

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase.

Freeze-thawed rat liver mitochondria were extensively washed with potassium phosphate, pH 7.5, and the residue was extracted with 10 mM potassium phosphate, pH 7.5, 1% (w/v) sodium cholate, 0.5 M KCl. The four beta-oxidation enzyme activities of the washes and the last extract were assayed with substrates of various carbon chain lengths. Our data suggest that the last extract contains a novel acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase. A novel acyl-CoA dehydrogenase was purified. The molecular masses of the native enzyme and the subunit were estimated to be 150 and 71 kDa, respectively. One mole of enzyme contained 2 mole of FAD. These properties and immunochemical properties of the enzyme differed from those of three other acyl-CoA dehydrogenases: short-, medium-, and long-chain acyl-CoA dehydrogenases. Carbon chain length specificity of the enzyme differed from that of other acyl-CoA dehydrogenases. The enzyme was active toward CoA esters of long- and very-long-chain fatty acids, but not toward those of medium- and short-chain fatty acids. The specific enzyme activity was greater than 10 times that of long-chain acyl-CoA dehydrogenase when palmitoyl-CoA was used as substrate. We propose the name "very-long-chain acyl-CoA dehydrogenase" for this enzyme.     

Essential disulfide and sulfhydryl groups for organic cation transport in renal brush-border membranes

The disulfide reducing agent, dithiothreitol (DTT) and the sulfhydryl-modifying reagents p-chloromercuribenzenesulfonic acid and N-ethylmaleimide (NEM) were employed to assess the role of disulfide and sulfhydryl groups in organic cation transport. The transport of N1-[3H]methylnicotinamide (NMN), a prototypic organic cation, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. DTT inhibited NMN transport reversibly with an IC50 of 250 microM/mg of protein. 5 mM NMN protected against DTT inactivation. The specificity of substrate protection was demonstrated by showing that D-glucose had no effect on the DTT inactivation of NMN transport and conversely that NMN had no effect on the DTT inactivation of D-glucose transport. Disulfide bonds reduced by DTT could be reoxidized by washing with excess buffer or by addition of 0.02% H2O2 thereby restoring NMN transport. p-Chloromercuribenzenesulfonic acid reversibly inactivated NMN transport with an IC50 of 25 microM/mg of protein. 5mM NMN protected against inactivation. NEM irreversibly inactivated transport with an IC50 of 250 microM/mg of protein. The rate of NMN inactivation by NEM followed pseudo-first order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 1.3. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of NEM inactivates 1 sulfhydryl group/active transport unit. The presence of 5 mM NMN affected the rate of NEM (2.5 mM) inactivation: the t1/2 values for inactivation in the presence and absence of substrate were 7.3 and 2.0 min, respectively. The results demonstrate an essential requirement for disulfide and sulfhydryl groups.     

Sulfhydryl groups of an extramitochondrial acetyl-CoA hydrolase from rat liver

An extramitochondrial acetyl-CoA hydrolase (EC 3.1.2.1) purified from rat liver was inactivated by heavy metal cations (Hg2+, Cu2+, Cd2+ and Zn2+), which are known to be highly reactive with sulfhydryl groups. Their order of potency for enzyme inactivation was Hg2+ greater than Cu2+ greater than Cd2+ greater than Zn2+. This enzyme was also inactivated by various sulfhydryl-blocking reagents such as p-hydroxymercuribenzoate (PHMB), N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and iodoacetate (IAA). DL-Dithiothreitol (DTT) reversed the inactivation of this enzyme by DTNB markedly, and that by PHMB slightly, but did not reverse the inactivations by NEM, DTNB and IAA. Benzoyl-CoA (a substrate-like competitive inhibitor) and ATP (an activator) greatly protected acetyl-CoA hydrolase from inactivation by PHMB, NEM, DTNB and IAA. These results suggest that the essential sulfhydryl groups are on or near the substrate binding site and nucleotide binding site. The enzyme contained about four sulfhydryl groups per mol of monomer, as estimated with DTNB. When the enzyme was denatured by 4 M guanidine-HCl, about seven sulfhydryl groups per mol of monomer reacted with DTNB. Two of the four sulfhydryl groups of the subunit of the native enzyme reacted with DTNB first without any significant inactivation of the enzyme, but its subsequent reaction with the other two sulfhydryl groups seemed to be involved in the inactivation process.     

A sulfhydryl presumed essential is not required for catalysis by an aminoacyl-tRNA synthetase

Several aminoacyl-tRNA synthetases are sensitive to reagents that modify sulfhydryl groups. We report here the significance of N-ethylmaleimide (NEM)-mediated inactivation of Escherichia coli glycyl-tRNA synthetase, and alpha 2 beta 2 enzyme. We confirmed earlier observations that NEM abolishes synthetase-catalyzed aminoacylation with pseudo-first order kinetics and provided a second method of proof that the site of inactivation is located in the beta-subunit. Using oligonucleotide-directed mutagenesis of the glyS gene, each beta-subunit cysteine codon (positions 98, 395, and 450) was replaced, individually, by an alanine codon. The three resulting mutant proteins are each active in vivo, and their in vitro aminoacylation activities are comparable to that of the native enzyme. A mutant incorporating all three amino acid substitutions is also active in vivo and in vitro. These results establish conclusively that a beta-subunit cysteine thiol is not required for the catalysis of aminoacylation. The Cys98----Ala and Cys450----Ala mutants are inactivated by NEM with the same kinetics as the wild-type protein. However, the Cys395----Ala mutant is refractory to NEM. This suggests that NEM inactivation of the native enzyme is due to alkylation of Cys395. Aware that inactivation may result from steric effects, we constructed a mutant with a bulkier amino acid residue at position 395 (Cys395----Gln). The aminoacylation activity of this protein is less than 10% of that of the wild-type enzyme. The glutamine substitution affects only the tRNA-dependent step of the reaction--the rate of glycyl adenylate synthesis is not lowered. In these features, the mutant resembles the NEM-inactivated protein. We propose that the NEM sensitivity of glycyl-tRNA synthetase, and possibly of other synthetases, arises from steric or conformational effects of the alkylated cysteine side chain.     

Sulfhydryl groups are essential for organic cation exchange in rabbit renal basolateral membrane vesicles

The effect of N-ethylmaleimide (NEM), an irreversible sulfhydryl modifying reagent, on the transport of organic cations in the renal basolateral membrane was examined. The studies were conducted examining the exchange of [3H]tetraethylammonium (TEA) for unlabeled TEA in basolateral membrane vesicles isolated from the outer cortex of rabbit kidneys. NEM inactivated TEA transport in a dose-dependent fashion with an IC50 value of 260 microM. The rate of TEA transport inactivation followed apparent pseudo-first-order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 4.0. The data imply that inactivation involves the binding of at least four molecules of NEM per active transport unit. This is most consistent with the presence of four sulfhydryl groups at this site. The substrate TEA displayed a dose-dependent enhancement of NEM inactivation, with 50% enhancement occurring at 365 microM TEA. Another organic cation, N1-methylnicotinamide, known to share a common transport mechanism with the TEA/TEA exchanger is also capable of increasing the reactivity of sulfhydryl groups to NEM. These results demonstrate that there are essential sulfhydryl groups for organic cation transport in the basolateral membrane. In addition, the capability of organic cations to alter the susceptibility to sulfhydryl modification suggests that these groups may have a dynamic role in the transport process.     

The reactivity of sulfhydryl groups of yeast DNA dependent RNA polymerase I

The number of reactive cysteine residues of yeast RNA polymerase I was determined and their function was studied using parachloromercury benzoate (pCMB), dithiobisnitrobenzoate (DTNB) and N-ethyl-maleimide (NEM) as modifying agents. By treatment with DTNB about 45 sulfhydryl groups react in the presence of 8M urea. Under non-denaturing conditions only 20 sulfhydryl groups are reactive with pCMB and DTNB. Both reagents completely inactivate the enzyme and this effect can be reversed by reducing agents. The sedimentation coefficient and the subunit composition are not affected when the enzyme is inactivated. Two of the most reactive sulfhydryl groups are necessary for activity. The modification of these groups is partially protected by substrates and DNA, suggesting that they are involved either in catalysis or in the maintenance of the conformation of the active site. Experiments with 14C-NEM indicate that the most reactive groups are located in subunits of 185,000, 137,000 and 41,000 daltons.     

Inactivation of an invertebrate acetylcholinesterase by sulfhydryl reagents: a reconsideration of the implications for insecticide design

Previously we used site-directed mutagenesis, in vitro expression, and molecular modeling to investigate the inactivation of an invertebrate acetylcholinesterase, cholinesterase 2 from amphioxus, by the sulfhydryl reagents 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). We created the mutants C310A, C466A, C310A/C466A and C310A/F312I to assess the roles of the two cysteines and a proposal that the increased rate of inactivation previously found in an F312I mutant was due to increased access of sulfhydryl reagents to Cys310. Our results indicated that both of the cysteines could be involved in inactivation by sulfhydryl reagents, but that the cysteine near the acyl pocket was more accessible. We speculated that the inactivation of aphid AChEs by sulfhydryl reagents was due to the presence of a cysteine homologous to Cys310 and proposed that this residue could be a target for a specific insecticide. Here we reconsider this proposal.     

Differential sensitivities of the subunits of mammalian ribonucleotide reductase to proteases, sulfhydryl reagents, and heat

Ribonucleotide reductase catalyzes the rate-limiting step in the formation of 2'-deoxyribonucleoside 5'-triphosphates. It consists of two nonidentical protein subunits, the nonheme iron subunit, and the effector-binding subunit. It has previously been shown that these two components making up the active enzyme species are not coordinately synthesized or degraded. It was found that the effector-binding subunit was more sensitive to proteolysis by chymotrypsin, to heating at 55 degrees C, and to the sulfhydryl reagents, pCMB and NEM. The nonheme iron subunit was more sensitive to trypsin treatment. ATP and dATP protected the effector-binding subunit from proteolytic inactivation. Neither ATP nor CDP protected the effector-binding subunit from inactivation by the sulfhydryl reagents. These data indicate that the protein properties of the two subunits of mammalian ribonucleotide reductase are significantly different.     

Inactivation of pig heart NADP-specific isocitrate dehydrogenase by two affinity reagents is due to reaction with a cysteine not essential for function.

Pig heart NADP-dependent isocitrate dehydrogenase is 65% inactivated by 3-bromo-2-ketoglutarate (Ehrlich, R.S., and Colman, R.F., 1987, J. Biol. Chem. 262, 12,614-12,619) and 90% inactivated by 2-(4-bromo-2,3-dioxobutylthio)-1,N6- ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) (Bailey, J.M., and Colman, R.F., 1987, J. Biol. Chem. 262, 12,620-12,626). Both inactivation reactions result in enzyme with an incorporation of 1.0 mol reagent/mol enzyme dimer and both modified enzymes bind only 1.0 mol manganous isocitrate or NADPH/mol enzyme dimer as compared to 2.0 mol manganous isocitrate or NADPH/mol enzyme dimer for unmodified enzyme. The inactivation reactions, which occur at or near the nucleotide binding site, are mutually exclusive. Reaction with either affinity reagent led to the isolation of the same modified triskaidekapeptide, DLAGXIHGLSNVK. We have isolated from isocitrate dehydrogenase a peptide, DLAGCIHGLSNVK, that had been modified by N-ethylmaleimide (NEM) with no loss of enzymatic activity. We now show that enzyme modified by NEM in the presence of isocitrate plus Mn2+ retains full catalytic activity but is not inactivated by either of the affinity reagents; thus, all three reagents appear to react at the same site. The analysis of HPLC tryptic maps of isocitrate dehydrogenase treated under denaturing conditions with iodo[3H]acetic acid or [3H]NEM demonstrates that both bromoketoglutarate and 2-BDB-T epsilon A-2',5'-DP react with the cysteine residue of DLAGCIHGLSNVK. We conclude that the cysteine of this triskaidekapeptide is close to the coenzyme binding site but is not essential for catalytic function.     

Effect of cysteine residues on the activity of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA synthetase (ArgRS) from Escherichia coli (E. coli) contains four cysteine residues. In this study, the role of cysteine residues in the enzyme has been investigated by chemical modification and site-directed mutagenesis. Titration of sulfhydryl groups in ArgRS by 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) suggested that a disulfide bond was not formed in the enzyme and that, in the native condition, two DTNB-sensitive cysteine residues were located on the surface of ArgRS, while the other two were buried inside. Chemical modification of the native enzyme by iodoacetamide (IAA) affected only one DTNB-sensitive cysteine residue and resulted in 50% loss of enzyme activity, while modification by N-ethylmeimide (NEM) affected two DTNB-sensitive residues and caused a complete loss of activity. These results, when combined with substrate protection experiments, suggested that at least the two cysteine residues located on the surface of the molecule were directly involved in substrates binding and catalysis. However, changing Cys to Ala only resulted in slight loss of enzymatic activity and substrate binding, suggesting that these four cysteine residues in E. coli ArgRS were not essential to the enzymatic activity. Moreover, modifications of the mutant enzymes indicated that the two DTNB- and NEM-sensitive residues were Cys(320) and Cys(537) and the IAA-sensitive was Cys(320). Our study suggested that inactivation of E. coli ArgRS by sulfhydryl reagents is a result of steric hindrance in the enzyme.     

Chemical modification of rat hepatic microsomes with N-ethylmaleimide results in inactivation of both UDP-N-acetylglucosamine-dependent stimulation of glucuronidation and UDP-glucuronic acid uptake.

Chemical modification of rat hepatic microsomes with N-ethylmaleimide (NEM) resulted in inactivation of UDP-N-acetylglucosamine (UDP-GlcNAc)-dependent stimulation of glucuronidation of p-nitrophenol. Inactivation kinetics and pH dependence were in agreement with the modification of a single sulfhydryl group. NEM also inactivated the uptake of UDP-glucuronic acid (UDP-GlcUA) but not UDP-glucose. With various sulfhydryl-modifying reagents, the inactivation of UDP-GlcUA uptake was linked to that of glucuronidation. UDP-GlcUA protected against NEM-sensitive inactivation of both UDP-GlcNAc-dependent stimulation of glucuronidation and UDP-GlcUA uptake, suggesting that the sulfhydryl group is located within or near the UDP-GlcUA binding site of the microsomal protein involved in the stimulation. Using microsomes labeled with biotin-conjugated maleimide and immunopurification with anti-peptide antibody against UDP-glucuronosyltransferase family 1 (UGT1) isozymes, immunopurified UGT1s were found to be labeled with the maleimide and UDP-GlcUA protected against the labeling as it did with the NEM-sensitive inactivation. These data suggest the involvement of a sulfhydryl residue of microsomal protein in the UDP-GlcNAc-dependent stimulation mechanism via the stimulation of UDP-GlcUA uptake into microsomal vesicles.     

Biospecific inactivation of aspartase by L-aspartic-β-semialdehyde

Aspartase purified from Escherichia coli W cells was rapidly and irreversibly inactivated by L-aspartic-β-semialdehyde (ASA), a substrate analog, following pseudo-first order kinetics. The inactivation rate showed a tendency to saturate as the ASA concentration increased. The increase in pH and the addition of Mg²⁺ at the alkaline pH accelerated the inactivation. In addition to chemically synthesized ASA, modification of aspartase by enzymatically generated ASA was attempted. Since the reaction equilibrium of homoserine dehydrogenase is extremely unfavorable for ASA formation, glutamate dehydrogenase reaction was coupled to it. When aspartase was incubated with these two enzyme systems, a time-dependent inactivation was observed. L-Aspartate, a substrate for the enzyme, protected it from inactivation. Analysis of the sulfhydryl group indicated that among 9 sulfhydryl groups per enzyme subunit, one residue essential for the activity was involved in the ASA-mediated inactivation.     

5,5′-Dithiobis-(2-Nitrobenzoic Acid) as a Probe for a Non-Essential Cysteine Residue at the Medium Chain Acyl-Coenzyme a Dehydrogenase Binding Site of the Human ‘Electron Transferring Flavoprotein’ (ETF)

Abstract

Human ‘electron transferring flavoprotein’ (ETF) was inactivated by the thiol-specific reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The kinetic profile showed the reaction followed pseudo-first-order kinetics during the initial phase of inactivation. Monitoring the release of 5-thio-2-nitrobenzoate (TNB) showed that modification of 1 cysteine residue was responsible for the loss of activity. The inactivation of ETF by DTNB could be reversed upon incubation with thiol-containing reagents. The loss of activity was prevented by the inclusion of medium chain acyl-CoA dehydrogenase (MCAD) and octanoyl-CoA. Cyanolysis of the DTNB modified-ETF with KCN led to the release of TNB accompanied presumably by the formation of the thio-cyano enzyme and with almost full recovery of activity. Conservation studies and the lack of 100% inactivation, however, suggested that this cysteine residue is not essential for the interaction with MCAD.     

Inactivation of protein disulfide isomerase by alkylators including alpha,beta-unsaturated aldehydes at low physiological pHs

Protein disulfide isomerase (PDI) is known to contain the thioredoxin box motif with a low pKa cysteine residue. To investigate the reactivity of PDI with thiol modifiers at low physiological pHs, either the reduced (PDIred) or oxidized form (PDIoxid) of PDI was exposed to various alkylating ragents. When PDI was incubated with iodoacetamide at pH 6.3 for 30 min at 38 degrees C, a remarkable inactivation (>90%) of PDIred was caused by iodoacetamide (IC50=8 microM). However, PDIoxid was only slightly inactivated (approximately 18%) by iodoacetamide. Similarly, PDIred was significantly inactivated by N-ethylmaleimide (NEM), but PDIoxid was not. When the inactivation by these alkylators was analyzed by pseudo-first order kinetics, NEM (k3=1.75x10(-2) s(-1); K(i)=124 microM) was observed to be more potent than iodoacetamide (k3=9.1x10(-3) s(-1); K(i)=311 microM). Interestingly, the inactivation of PDIred by iodoacetamide was greater at pH 6.3 than pH 7.0, in contrast to a similar inactivation potency of NEM at both pHs. Moreover, the maximal inactivation of PDIred or PDIoxid by iodoacetamide was mainly observed around pH 6.0. In addition, PDIred was found to be inactivated by acrolein (IC50=10 microM) at pH 6.3, and this inactivation was also greater at pH 6.3 than at pH 7. Based on these results, we suggest that PDIred is susceptible to inactivation by alkylators including endogenous alpha,beta-unsaturated aldehydes at low physiological pHs.     

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. The inactivation by mono and bifunctional maleimides was partially prevented by Mg²⁺ and l-malate, and NADP prevented the inactivation almost totally. Determination of the number of reactive sulfhydryl groups of the native enzyme with [³H]NEM in the absence or presence of NADP showed that inactivation occurred concomitantly with the modification of two cysteinyl residues per enzyme monomer. The presence of these two essential residues was confirmed by titration of sulfhydryl groups with [³H]NEM in the enzyme previously modified by o-phenylenebismaleimide in the absence or presence of NADP.     

A cysteine residue (cysteine-116) in the histidinol binding site of histidinol dehydrogenase

Salmonella typhimurium L-histidinol dehydrogenase (EC 1.1.1.23), a four-electron dehydrogenase, was inactivated by an active-site-directed modification reagent, 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl). The inactivation followed pseudo-first-order kinetics and was prevented by low concentrations of the substrate L-histidinol or by the competitive inhibitors histamine and imidazole. The observed rate saturation kinetics for inactivation suggest that NBD-Cl binds to the enzyme noncovalently before covalent inactivation occurs. The UV spectrum of the inactivated enzyme showed a peak at 420 nm, indicative of sulfhydryl modification. Stoichiometry experiments indicated that full inactivation was correlated with modification of 1.5 sulfhydryl groups per subunit of enzyme. By use of a substrate protection scheme, it was shown that 0.5 sulfhydryl per enzyme subunit was neither protected against NBD-Cl modification by L-histidinol nor essential for activity. Modification of the additional 1.0 sulfhydryl caused complete loss of enzyme activity and was prevented by L-histidinol. Pepsin digestion of NBD-modified enzyme was used to prepare labeled peptides under conditions that prevented migration of the NBD group. HPLC purification of the peptides was monitored at 420 nm, which is highly selective for NBD-labeled cysteine residues. By amino acid sequencing of the major peptides, it was shown that the reagent modified primarily Cys-116 and Cys-377 and that the presence of L-histidinol gave significant protection of Cys-116. The presence of a cysteine residue in the histidinol binding site is consistent with models in which formation and subsequent oxidation of a thiohemiacetal occurs as an intermediate step in the overall reaction.     

Mechanism-based inactivation of human glutaryl-CoA dehydrogenase by 2-pentynoyl-CoA: rationale for enhanced reactivity

2-Pentynoyl-CoA inactivates glutaryl-CoA dehydrogenase at a rate that considerably exceeds the rates of inactivation of short chain and medium chain acyl-CoA dehydrogenases by this inhibitor and related 2-alkynoyl-CoAs. To determine the rate of inactivation by 2-pentynoyl-CoA, we investigated the inactivation in the presence of a non-oxidizable analog, 3-thiaglutaryl-CoA, which competes for the binding site. The enhanced rate of inactivation does not reflect an alteration in specificity for the acyl group, nor does it reflect the covalent modification of a residue other than the active site glutamate. In addition to determining the inactivation of catalytic activity a spectral intermediate was detected by stopped-flow spectrophotometry, and the rate constants of formation and decay of this charge transfer complex (lambdamax approximately 790 nm) were determined by global analysis. Although the rate-limiting step in the inactivation of the other acyl-CoA dehydrogenases can involve the abstraction of a proton at C-4, this is not the case with glutaryl-CoA dehydrogenase. Glutaryl-CoA dehydrogenase is also differentiated from other acyl-CoA dehydrogenases in that the catalytic base must access both C-2 and C-4 in the normal catalytic pathway. Access to C-4 is not obligatory for the other dehydrogenases. Analysis of the distance from the closest carboxylate oxygen of the glutamate base catalyst to C-4 of a bound acyl-CoA ligand for medium chain, short chain, and isovaleryl-CoA dehydrogenases suggests that the increased rate of inactivation reflects the carboxylate oxygen to ligand C-4 distance in the binary complexes. This distance for wild type glutaryl-CoA dehydrogenase is not known. Comparison of the rate constants of inactivation and formation of a spectral species between wild type glutaryl-CoA dehydrogenase and a E370D mutant are consistent with the idea that this distance in glutaryl-CoA dehydrogenase contributes to the enhanced rate of inactivation and the 1,3-prototropic shift catalyzed by the enzyme.     

Selective inactivation of various acyl-CoA dehydrogenases by (methylenecyclopropyl)acetyl-CoA

Inactivation of five distinct acyl-CoA dehydrogenases by (methylenecyclopropyl)acetyl-CoA (MCPA-CoA), the toxic metabolite of hypoglycin from unripe ackee fruit, was investigated using purified enzyme preparations. Short-chain acyl-CoA (SCADH), medium-chain acyl-CoA (MCADH) and isovaleryl-CoA (IVDH) dehydrogenases were severely and irreversibly inactivated by MCPA-CoA, while 2-methyl-branched chain acyl-CoA dehydrogenase (2-meBCADH) was only slowly and mildly inactivated. Long-chain acyl-CoA dehydrogenase (LCADH) was not significantly inactivated, even after prolonged incubation with MCPA-CoA. Inactivation of SCADH, MCADH and IVDH was effectively prevented by the addition of substrate. This mode of inactivation by MCPA-CoA explains the urinary metabolite profile in hypoglycin treated-rats, which includes large amounts of metabolites from fatty acids and leucine, and relatively small amounts of those from valine and isoleucine. Spectrophotometric titration of SCADH and MCADH with MCPA-CoA, together with the protective effects of substrate, indicates that MCPA-CoA is acted upon by, and exerts in turn irreversible inactivation of, SCADH and MCADH, confirming that MCPA-CoA is a suicide inhibitor (Wenz et al. (1981) J. Biol. Chem. 256, 9809-9812). Spectrophotometric titration data of LCADH and MCPA-CoA is typical of non-reacting CoA ester.