Inhibition and alternate-substrate studies on the mechanism of malic enzyme

A number of dead-end inhibitors and alternate substrates were examined to gain an understanding of the substrate specificity and mechanism of malic enzyme. Comparison of Ki values for competitive inhibitors suggested that binding of the l-carboxyl of L-malate is by ion pairing with lysine or arginine, while binding of the 4-carboxyl is weaker, and probably of the induced-dipolar type. The 2-hydroxyl hydrogen bonds to a catalytic group, which, when it is protonated, adsorbs the keto form of oxalacetate. Since the only molecule other than L-malate that is oxidized is L-malate-beta-amide, carbon 4 must be trigonal for substrate activity, although a tetrahedral carbon bearing one or two hydroxyl groups gives good binding. Hydroxy groups at carbon 3 contribute to binding, but prevent substrate activity. Hydroxy and ketomalonates are bound more strongly than any of the four carbon acids, suggesting that the latter are bound with some strain. In inhibition studies, pyruvate analogues were competitive vs. pyruvate but noncompetitive vs. malate, while malate analogues were competitive vs. malate and noncompetitive vs. pyruvate. These compounds thus bind to both enzyme-triphosphopyridine nucleotide (E-TPN) and enzyme-reduced triphosphopyridine nucleotide (E-TPNH), but only malate analogues prevent release of TPN, while pyruvate analogues prevent release of TPNH. Ketomalonate and oxalacetate, both of which are slowly reduced by the enzyme in the presence of TPNH and thus must combine in the keto form with E-TPNH,, appear to combine with E-TPN mainly in the gem-diol (or for oxalacetate, also the enol) form. The substrate for the decarboxylation of oxalacetate at pH 4.5 is the keto form.     

Role of cysteines in the activation and inactivation of brewers' yeast pyruvate decarboxylase investigated with a PDC1-PDC6 fusion protein

Possible roles of the Cys side chains in the activation and inactivation mechanisms of brewers' yeast pyruvate decarboxylase were investigated by comparing the behavior of the tetrameric enzyme pdc1 containing four cysteines/subunit (positions 69, 152, 221, and 222) with that of a fusion enzyme (pdc1-6, a result of spontaneous gene fusion between PDC1 and PDC6 genes) that is 84% identical in sequence with pdc1 and has only Cys221 (the other three Cys being replaced by aliphatic side chains). The two forms of the enzyme are rather similar so far as steady-state kinetic parameters and substrate activation are considered, as tested for activation by the substrate surrogate pyruvamide. Therefore, if a cysteine is responsible for substrate activation, it must be Cys221. The inactivation of the two enzymes was tested with several inhibitors. Methylmethanethiol sulfonate, a broad spectrum sulfhydryl reagent, could substantially inactivate both enzymes, but was slightly less effective toward the fusion enzyme. (p-Nitrobenzoyl)formic acid is an excellent alternate substrate, whose decarboxylation product p-nitrobenzaldehyde inhibited both enzymes possibly at a Cys221, the only one still present in the fusion enzyme. Exposure of the fusion enzyme, just as of pdc1, to (E)-2-oxo-4-phenyl-3-butenoic acid type inhibitors/alternate substrates enabled detection of the enzyme-bound enamine intermediate at 440 nm. However, unlike pdc1, the fusion enzyme was not irreversibly inactivated by these substrates. These substrates are now known to cause inactivation of pdc1 with concomitant modification of one Cys of the four [Zeng, X.; Chung, A.; Haran, M.; Jordan, F. (1991) J. Am. Chem. Soc. 113, 5842-49].(ABSTRACT TRUNCATED AT 250 WORDS)     

NADP-malic enzyme from maize leaves: a fluorescence study

NADP-malic enzyme from maize leaves is covalently labeled with a fluorescent-SH reactive probe eosin-5-maleimide (EMA), which reacts with groups that are totally protected by NADP against inactivation. The comparison of the emission fluorescence spectra of the native and the modified enzyme suggests the proximity of the fluorescent groups of the native enzyme (probably tryptophanyl groups) and the EMA modified residues. Intrinsic fluorescence quenching studies shows that NADP is the only substrate capable to interact with the fluorescent excited groups of the enzyme, while Mg2+ is able to increase this interaction. Quenching studies of EMA-bound fluorescence shows that the NADP-binding site was modified and thus uncapable of further interaction with the nucleotide. When the results of protection studies are combined with those of extrinsic quenching experiments, we must conclude that EMA reacts with sulfhydryl groups that are involved in the NADP-binding site of the enzyme.     

Exponential heating in drug stability experiment and statistical evaluation of nonisothermal and isothermal prediction

A concerted conformational change in Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was evident from previous fluorescence on the quenching of the single Trp residue Trp-92 in the 4FTrp-AMP complexed enzyme. In this study, chemical modifications of the B. subtilis TrpRS were employed to further characterize this conformational change, with the single Trp residue serving as a marker for monitoring the change. Modifications of the enzyme by means of the Trp-specific agent N-bromosuccinimide (NBS) or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BNPS-skatole) inactivated the enzyme in accord with the essential role of Trp-92, as identified previously by site-directed mutagenesis. ATP sensitized TrpRS toward inactivation by NBS and BNPS-skatole, which suggested a conformational change that resulted in greater accessibility of Trp-92 toward modifications. In contrast, the cognate tRNATrp substrate exerted a specific protective effect against inactivation by both of the reagents, indicating that the TrpRS-tRNATrp interaction reduces the accessibility of Trp-92 under our experimental conditions. By comparison, modification of sulfhydryl groups by means of iodoacetamide did not reduce TrpRS activity. Observations on Trp-specific modification and substrate protection effects are discussed in the context of the Bacillus stearothermophilus TrpRS crystal structure.     

High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis. Implications for substrate activation in pyruvate decarboxylases

The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 A resolution and refined to a crystallographic R-factor of 16.2% and Rfree of 19.7%. The subunit consists of three domains, all of the alpha/beta type. Two of the subunits form a tight dimer with an extensive interface area. The thiamin diphosphate binding site is located at the subunit-subunit interface, and the cofactor, bound in the V conformation, interacts with residues from the N-terminal domain of one subunit and the C-terminal domain of the second subunit. The 2-fold symmetry generates the second thiamin diphosphate binding site in the dimer. Two of the dimers form a tightly packed tetramer with pseudo 222 symmetry. The interface area between the dimers is much larger in pyruvate decarboxylase from Z. mobilis than in the yeast enzyme, and structural differences in these parts result in a completely different packing of the subunits in the two enzymes. In contrast to other pyruvate decarboxylases, the enzyme from Z. mobilis is not subject to allosteric activation by the substrate. The tight packing of the dimers in the tetramer prevents large rearrangements in the quaternary structure as seen in the yeast enzyme and locks the enzyme in an activated conformation. The architecture of the cofactor binding site and the active site is similar in the two enzymes. However, the x-ray analysis reveals subtle but significant structural differences in the active site that might be responsible for variations in the biochemical properties in these enzymes.     

Identification of the nonsubstrate steroid binding site of rat liver glutathione S-transferase, isozyme 1-1, by the steroid affinity label, 3beta-(iodoacetoxy)dehydroisoandrosterone

3beta-(Iodoacetoxy)dehydroisoandrosterone (3beta-IDA), an analogue of the electrophilic substrate, Delta5-androstene-3,17-dione, as well as an analogue of several other steroid inhibitors of glutathione S-transferase, was tested as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent loss of enzyme activity is observed upon incubation of 3beta-IDA with the enzyme. The rate of enzyme inactivation exhibits a nonlinear dependence on 3beta-IDA concentration, yielding an apparent Ki of 21 microM. Upon complete inactivation of the enzyme, a reagent incorporation of approximately 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer is observed. Protection against inactivation and incorporation is afforded by alkyl glutathione derivatives and nonsubstrate steroid ligands such as 17beta-estradiol-3,17-disulfate but, surprisingly, not by Delta5-androstene-3,17-dione or any other electrophilic substrate analogues tested. These results suggest that the site of reaction is within the nonsubstrate steroid binding site of the enzyme, which is distinguishable from the electrophilic substrate binding site, near the active site of the enzyme. Two cysteine residues, Cys17 and Cys111, are modified in nearly equal amounts, despite an average reagent incorporation of 1 mol/mol enzyme subunit. Isolation of enzyme subunits indicates the presence of unmodified, singly labeled, and doubly labeled subunits, consistent with mutually exclusive modification of cysteine residues across enzyme subunits; i.e., modification of Cys111 on subunit A prevents modification of Cys111 on subunit B and similarly for Cys17. Molecular modeling analysis suggests that Cys17 and Cys111 are located in the nonsubstrate steroid binding site, within the cleft between the subunits of the dimeric enzyme.     

Inactivation of 3-hydroxy-3-methylglutaryl-CoA synthase and other Acyl-CoA-utilizing enzymes by 3-Oxobutylsulfoxyl-CoA

3-Oxobutylsulfoxyl-CoA has been produced by oxidation of S-3-oxobutyl-CoA, the thioether analog of acetoacetyl-CoA. Avian hydroxymethylglutaryl-CoA (HMG-CoA) synthase is inactivated by oxobutylsulfoxyl-CoA in a time-dependent fashion. Protection against inactivation is afforded by the substrate, acetyl-CoA, suggesting that inactivation involves modification of the enzyme's active site. Pretreatment of HMG-CoA synthase with the inactivator blocks the enzyme's ability to form Michaelis and acetyl-S-enzyme intermediates, supporting the hypothesis that modification is active-site directed. Incubation of enzyme with oxobutylsulfoxyl-[32P]CoA followed by precipitation with trichloroacetic acid indicates that inactivation correlates with stoichiometric formation of a covalent adduct between enzyme and a portion of the inactivator that includes the CoA nucleotide. The observation of reagent partitioning suggests that HMG-CoA synthase catalyzes conversion of oxobutylsulfoxyl-CoA into a reactive species that modifies the protein. Treatment of inactivated enzyme with DTT or other mercaptans restores enzyme activity and reverses the covalent modification with release of CoASH. Oxobutylsulfoxyl-CoA inactivates beta-ketothiolase and HMG-CoA lyase in a process that is also reversed by DTT. These three enzymes all contain active site cysteines, suggesting that inactivation results from disulfide formation between a cysteine and the CoA moiety of the inhibitor. The data are consistent with the hypothesis that enzymatic cleavage of oxobutylsulfoxyl-CoA results in the transient formation of a sulfenic acid derivative of CoA which subsequently reacts to form a stable disulfide linkage to protein.     

Mode of action of site-directed irreversible folate analogue inhibitors of thymidylate synthase

5,8-Dideazafolate analogues are tight binding but not irreversible inhibitors of thymidylate synthase (TS). However, when a chloroacetyl (ClAc) group is substituted at the N10-position of 2-desamino-2-methyl-5,8-dideazafolate (DMDDF), the resulting compound, ClAc-DMDDF, although still a reversible inhibitor (KI = 3.4 x 10(-3) M), gradually inactivates thyA-TS irreversibly at a rate of 0.37 min-1. The corresponding iodoacetyl derivative alkylated the enzyme somewhat slower (k3 = 0.15 min-1 ) than ClAc-DMDDF but was bound more tightly (KI = 1.4 x 10(-5) M), resulting in a second-order rate constant (k3/KI) of inactivation that was 100-fold greater than that of ClAc-DMDDF. A tryptic digest of the ClAc-DMDDF-inactivated enzyme yielded a peptide on HPLC, which revealed that cysteine-146, the residue at the active site that is intimately involved in the catalytic process, had reacted with ClAc-DMDDF to form a covalent bond. This derivative was confirmed indirectly by Edman analysis and more directly by mass spectrometry. Deoxyuridine 5'-monophosphate, a substrate in the catalytic reaction, protected against inactivation. Similar to previously described Lactobacillus casei TS inhibition studies with sulfhydryl reagents [Galivan, J., Noonan, J., and Maley, F. (1977) Arch. Biochem. Biophys. 184, 336-345], the kinetics of inhibition suggested that complete inhibition occurs on reaction of only one of the two active site cysteines, although sequence and amino acid analysis revealed that iodoacetate and ClAc-DMDDF had reacted with both active site cysteines. These studies demonstrate that a sulfhydryl reactive compound that is directed to the folate binding site of TS may diffuse to the active site cysteine, and form a covalent bond with this residue. How this inhibition comes about is suggested in a stereoscopic view of the ligand when modeled to the known crystal structure of Escherichia coli TS.     

Tryptophan residues in alpha-galactosidase from Trichoderma reesei

Tryptophan residues in alpha-galactosidase were modified with bromosuccinimide. The fact that galactose, a specific inhibitor of alpha-galactosidase, does not prevent this modification demonstrates that tryptophan residues are not located in galactose binding sites. Analysis of the inactivation kinetics revealed two groups of Trp residues (8.5 and 7.5 residues) with different accessibility for N-bromosuccinimide. We studied specific quenching of alpha-galactosidase fluorescence resulting from modification of an sulfhydryl group in the active site of the enzyme with Hg2+ and Ag+ ions. The specific quenching is due to conformational changes of the enzyme. Forster's radii were determined for various protein--chromophore complexes. Dynamic quenching of alpha-galactosidase fluorescence was investigated. To describe abnormal dynamic quenching in alpha-galactosidase, a modification of the Stern--Volmer equation is suggested.     

Histidine decarboxylase of Lactobacillus 30a: inactivation and active-site labeling by L-histidine methyl ester

Histidine decarboxylase from Lactobacillus 30a is rapidly and irreversibly inactivated upon incubation with L-histidine methyl ester. The rate of inactivation is first-order with respect to remaining active enzyme and exhibits saturation kinetics with a kinact of 1.2 mM and an apparent first-order rate constant of 0.346 min-1 at pH 4.8 and 25 degrees C. On complete inactivation, 3 mol of [14C]histidine (from L-[14C]histidine methyl ester) and 2 mol of 14C (from L-histidine [14C]methyl ester) are bound in nondialyzable form per mol (190 000 g) of protein inactivated with a corresponding loss of three of the five DTNB-titratable--SH groups that are essential for activity of the native enzyme. Imidazole propionate, a competitive inhibitor of the enzyme, protects against inactivation, loss of --SH groups, and incorporation of radioactivity from both the histidine and the methyl ester moieties of the labeled inhibitor, and kinetic evidence indicates that imidazole propionate and histidine methyl ester compete for binding at the active site of histidine decarboxylase in a mutually exclusive manner. Treatment of the labeled protein with either alkali or hydroxylamine results in the quantitative release of radioactivity. These data suggest that inactivation of histidine decarboxylase by L-histidine methyl ester results from two different modes of interaction between the inhibitor and the active site of histidine decarboxylase; the major interaction involves an essential -SH group.     

Hysteretic response of human erythrocyte pyruvate kinase to phosphoenolpyruvate. Potential role in regulation of 2,3-bisphosphoglycerate metabolism

Human erythrocyte pyruvate kinase (EC 2.7.1.40, ATP-pyruvate phosphotransferase) was found to display a time-dependent activation (lag phase) in the reaction progress curves. The extent of this lag phase depended upon the treatment of the enzyme prior to assay. Preincubation of the enzyme with adenine nucleotides amplified the lag, whereas prior treatment with phosphoenolpyruvate diminished it. The activation process was first order in enzyme with the pseudo first order rate constants being a hyperbolic function of phosphoenolpyruvate concentration. The data provide evidence for a phosphoenol-pyruvate-mediated conversion of the enzyme to a more active form. Studies with the irreversible sulfhydryl inhibitor, N-ethylmaleimide (MalNEt), provided additional evidence for different conformational states of the enzyme induced by its substrates and effectors. Adenine nucleotides were found to promote inactivation by MalNEt and phosphoenolpyruvate protected against MalNEt. The possible metabolic significance of this "hysteretic" pyruvate kinase is discussed in relation to the known role of this enzyme in 2,3-bisphosphoglycerate metabolism (Rose, I.A. (1971) Exp. Eye Res. 11, 264-272).     

Inactivation of D-3-hydroxybutyrate dehydrogenase by fumaroyl bis(methyl phosphate)

Fumaroyl bis(methyl phosphate) reacts with the NADH-dependent enzyme, D-3-hydroxybutyrate dehydrogenase, leading to irreversible inactivation. The bifunctional reagent cross-links the subunits of the enzyme. The inactivation is subject to saturation and protection by substrate, consistent with the reaction occurring at the active site. The stoichiometry of inactivation indicates two active sites undergo reaction with each equivalent of reagent. These results indicate that the dimeric enzyme has contiguous active sites. The reagent is likely to react with an active site lysine, consistent with previous suggestions.     

Identification of the subunit and important target peptides of pig heart NAD-dependent isocitrate dehydrogenase modified by the affinity label adenosine 5'-O-[S-(4-bromo-2, 3-dioxobutyl)thiophosphate]

Pig heart NAD-dependent isocitrate dehydrogenase is inactivated by adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate] (AMPS-BDB) with incorporation of 1.78 mol of reagent/mol of average subunit. Complete protection against the inactivation is provided by 20 mM isocitrate + 1 mM Mn2+, and the incorporation is decreased to about 1.3 mol of reagent/mol of average subunit. The addition of NAD, NADH, or Mn2+ alone has little effect on the functional changes produced by AMPS-BDB, while ADP gives only partial protection against the inactivation. The ability of ADP to decrease the Km for isocitrate is not affected by the AMPS-BDB modification of the enzyme. These results indicate that the isocitrate substrate site is the target of AMPS-BDB. The enzyme has three types of subunits with a tetramer having the composition alpha2 beta gamma. Here, [2-3H]AMPS-BDB-modified subunits are separated by HPLC on a C4 reverse-phase column, after the treatment of the modified enzyme with 4 M urea. The predominant radioactivity is distributed in alpha and gamma subunits. However, evidence based on recombination of subunits from modified and unmodified enzymes indicates that only labeling of the alpha subunit is responsible for inactivation by AMPS-BDB. Subsequently, the separated modified subunits were chemically cleaved by CNBr and then purified by HPLC using a C18 column. The labeled peptides were further digested by pepsin, purified by HPLC, and sequenced. These results indicate that R88 and R98 from the alpha subunit are the major targets of AMPS-BDB which cause inactivation and that these are at or near the isocitrate site of the enzyme.     

Purification and some properties of a xylanase from Aspergillus sydowii MG49

Aspergillus sydowii MG49 produces a 30-kDa exosplitting xylobiohydrolase during growth on xylan. A specific chemical modification and substrate protection analysis of purified xylanase provided evidence that tryptophan and carboxy and amino groups are present at the catalytic site of this enzyme. Thermal inactivation of the xylanase occurs because of irreversible polymolecular aggregation, which is slower in the presence of glycerol.     

Mechanism-based inactivation of serine transhydroxymethylases by D-fluoroalanine and related amino acids

Serine transhydroxymethylase, from lamb or rabbit liver, is known to catalyze slow transamination of D-alanine, but not of L-amino acids, in a tetrahydrofolate-independent reaction. Both enzymes will process the D-isomer of beta-fluoroalanine for alpha, beta-elimination of HF to yield an aminoacrylate-pyridoxal-P-enzyme intermediate. This intermediate partitions between harmless hydrolysis to pyruvate, NH4+, and active enzyme-pyridoxal-P (catalytic turnover) and suicidal enzyme alkylation by covalent modification with an average partition ratio of 40-60 turnovers/inactivation event/monomer unit of this tetrameric enzyme. Enzyme inactivation occurs with stoichiometric incorporation of radioactive label from D-[1,2-14C]fluoroalanine. Titration of enzymic cysteinyl --SH groups with 5,5'-dithiobis(2-nitrobenzoate) indicates loss of 1 --SH group on inactivation. Acid hydrolysis of radioactive-inactive enzyme confirms cysteine residue modification. Treatment of inactive enzyme with 6 M urea, then KBH4, followed by acid hydrolysis yields two radioactive compounds, lanthionine and S-carboxyhydroxyethylcysteine, in about equal amounts. The addition of tetrahydrofolate stimulates both pyruvate production and inactivation to equal extents with about a 200-fold rate acceleration at 0.5 mM tetrahydrofolate to turnover numbers of approximately 120 min-1. The Km for D-fluoroalanine is high, 10-60 mM, and this low substrate affinity suggests D-fluoroalanine will not be a useful in vivo agent for selective inactivation of liver cell serine transhydroxymethylases.     

Reactive oxygen species-mediated inactivation of pyruvate dehydrogenase

Brain ischemia reperfusion causes increased formation of reactive oxygen species (ROS). Activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH) has been shown to undergo a significant decrease following reperfusion of the ischemic tissue. We have examined the effect of a superoxide radical-generating system (xanthine oxidase/hypoxanthine, XO/HX) on the activity of this enzyme. Incubation of PDH in the presence of XO/HX resulted in its inactivation. The degree of the inactivation was dependent on the amount of XO present, which correlated linearly with the concentration of superoxide radical generated by this system. The activity of lactate dehydrogenase, an enzyme resistant to inactivation by ischemia reperfusion, was not affected by this system. Superoxide dismutase partially prevented and catalase exerted a nearly complete protective effect against the inactivation of PDH. Deferoxamine was partially protective. The sulfhydryl protective reagents, dithiothreitol and glutathione, prevented the inactivation of PDH, even though to varying degrees, which implicates sulfhydryl oxidation. A hydroxyl radical-generating system (hydrogen peroxide irradiated with ultraviolet radiation) effectively inactivated PDH. These results demonstrate that PDH is susceptible to damage and inactivation by ROS and point to the involvement of Fenton chemistry and hydroxyl radicals formed through it in PDH inactivation by XO/HX. A similar mechanism may be responsible for the PDH inactivation during ischemia/reperfusion.     

Inactivation of bovine liver 2-keto-4-hydroxyglutarate aldolase by cyanide in the presence of aldehydes

Kinetic data show that the irreversible inactivation of liver 2-keto-4-hydroxyglutarate aldolase observed when the enzyme is incubated with an aldehydic substrate (or substrate analogue) in the presence of cyanide is a biphasic process and can, under certain conditions, involve a direct interaction between the enzyme and cyanide. The kinetic data are consistent with a scheme consisting of three competing reactions: (1) irreversible addition of cyanide to the enzyme-substrate Schiff base intermediate, (2) reversible cyanohydrin formation between cyanide and the aldehydic substrate (or substrate analogue), and (3) an interaction of cyanide with the enzyme which is not substrate dependent. Approximately 0.4 mol of cyanide is associated with 1 mol (120 000 g) of enzyme when 2-keto-4-hydroxyglutarate aldolase is incubated with [14-C]-cyanide followed by exhaustive dialysis; an ionic attachment possibly at a carboxylate binding site, is suggested. Whereas native enzyme, not treated with cyanide, has ten Nbs2-titratable sulfhydryl groups, approximately one less such group reacts with Nbs2 when the aldolase is incubated with cyanide (in the absence of aldehydic substrate). It is suggested that the binding of cyanide results in a conformational change of the enzyme; conformational changes in the presence of cyanide are confirmed by circular dichroism spectra.     

Diagnosing depression in the medically ill: validity of a lay-administered structured diagnostic interview

N-Acetyltransferase (NAT), responsible for bioactivation and detoxification of arylamines, has been demonstrated to be widely distributed in many organisms ranging from humans to microorganisms. Using high performance liquid chromatography (HPLC) to analyze NAT activity in bacteria, the authors found that Pseudomonas aeruginosa exhibited high NAT activity with 2-aminofluorene (2-AF) as substrate. Characteristics of this bacterial NAT were further investigated. The N-acetylation catalyzed by this enzyme is an acetyl coenzyme A (AcCoA)-dependent reaction. As the concentration of AcCoA in the reaction mixture was increased, the apparent K(m) and Vmax for 2-AF increased. The K(m) and Vmax were 0.504 +/- 0.056 mM and 31.92 +/- 3.23 nmol/min/mg protein, respectively, for the acetylation of 2-AF with 0.5 mM AcCoA. The optimum pH for the enzyme activity was estimated to be around 8.5. It was active at a temperature range from 5 degrees C to 55 degrees C, with maximum activity at 37 degrees C. The enzyme activity was inhibited by divalent metal ions including Cu++, Fe++, Zn++, Ca++, Co++, Mn++, and Mg++, suggesting that a sulfhydryl group is involved in the N-acetylation activity. The three chemical modification agents, iodoacetamide, phenylglyoxal, and diethylpyrocarbonate, all exhibited a dose-, time-, and temperature-dependent inhibition effect. Preincubation of the NAT with AcCoA provided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhibition of phenylglyoxal. These results indicate that cysteine, histidine, and arginine residues are essential for this bacterial enzyme activity, and the first two are likely to reside on the AcCoA binding site, but arginine residue may be located only near the AcCoA binding site. Our data demonstrate that P. aeruginosa possesses highly active N-acetyltransferase which shares a similar catalytic mechanism as that of higher organisms. These findings are very helpful for further investigating the role of arylamine NAT in this bacterial species.     

Mutation of aspartate-233 to valine in mouse ornithine decarboxylase reduces enzyme activity

Ornithine decarboxylase is the first and key enzyme in mammalian polyamine biosynthesis. All eukaryotic ornithine decarboxylases contain several highly conserved regions and the amino acid residues 232-238 form one of the most highly conserved sequences. This region contains a glycine-rich sequence typically found in a number of pyridoxal 5'-phosphate-dependent or nucleotide-binding proteins. We mutated aspartate-233 which is the only acidic residue within this region to valine. This mutation causes striking sequence similarity with the guanine nucleotide binding domain of c-H-ras. Mutated ornithine decarboxylase cDNA with a mouse mammary tumor virus long terminal repeat promoter has been transfected for stable expression into ornithine decarboxylase-deficient C55.7 cells. Ornithine decarboxylase activity of the mutated enzyme was about 20% of wild-type ornithine decarboxylase activity and it was not activated by guanosine triphosphate like the ornithine decarboxylase isoform found in some tumors and rat brain. The mutation caused an increase in K(m) value of about 20-fold both for the substrate L-ornithine and for the cofactor pyridoxal 5'-phosphate. The Ki value for the irreversible inhibitor alpha-difluoromethylornithine was also increased, whereas the half-life of the enzyme was shortened. These results suggest that the region containing aspartate-233 is essential for binding of the cofactor and thus forms part of enzymatic active site, and the mutation of aspartate-233 to valine cannot, at least alone, cause the activation of ornithine decarboxylase by guanosine triphosphate (230).     

Increased UV exposure in Finland in 1993

N-Ethylmaleimide (NEM) inhibited the H(+)-ATPase (EC 3.6.1.35) from Kluyveromyces lactis with a second-rate constant of 200 M-1 min-1. H(+)-ATPase was partially protected by Mg-ADP. Low concentrations of Mg protected ATPase from the effects of NEM, while high Mg sensitized ATPase to NEM. The reaction of 14C-NEM with the native enzyme modified three cysteine residues/monomer, two of which were involved in 80% of the inactivation of the enzyme. In the presence of Mg-ADP, NEM binding to the first residue had only a slight effect on the activity (10-20% inhibition). After further incubation, the modification of a second cysteine residue (probably cys-221) inactivated the ATPase. Methyl methanethiosulfonate did not inhibit the H(+)-ATPase but resulted in a NEM-resistant H(+)-ATPase. There seems to be at least one cys (probably cys-532) at, or near, the nucleotide binding site of the H(+)-ATPase, which does not appear to be essential for activity. Modification of a second cys residue (cys-221) also resulted in inactivation by NEM; this residue was not protected by ADP and thus probably is not at the ATP binding site.