Stereospecificity of pantoyl lactone formed by yeast cells and purified yeast ketopantoyl lactone reductases.

Two highly purified yeast ketopantoyl lactone reductases form d-(?)-pantoyl lactone from ketopantoyl lactone, but whole or broken yeast forms a mixture of d-(?)- and l-(+)-pantoyl lactone. Of three potential routes for formation of l-(+)-pantoyl lactone, direct reduction of ketopantoyl lactone seems most likely.     

Microbial Reduction of Ketopantoyl Lactone to Pantoyl Lactone

The results of a microbial survey study have shown that the ability to reduce added ketopantoic acid (or ketopantoyl lactone) and accumulate pantoic acid (or pantoyl lactone) in the growth medium is widespread among diverse fungi. The reductions generally proceeded with less than full stereoselectivity. However, specific strains of the ascomycete Byssochlamys fulva were found to form D[-]-pantoic acid in unusually high yields and optical purity.     

One-Step Microbial Conversion of a Racemic Mixture of Pantoyl Lactone to Optically Active d-(-)-Pantoyl Lactone

Washed cells of Rhodococcus erythropolis IFO 12540 were found to convert only the l-(+)-isomer of pantoyl lactone to the d-(-)-isomer in a racemic mixture of pantoyl lactone. Under suitable reaction conditions, the amount of d-(-)-pantoyl lactone synthesized was 18.2 mg/ml (94.4% enantiomer excess; molar yield, 90.5%). This conversion was suggested to proceed through the following successive reactions: first, the enzymatic oxidation of l-(+)-pantoyl lactone to ketopantoyl lactone; second, the rapid and spontaneous hydrolysis of the ketopantoyl lactone to ketopantoic acid; and then, the enzymatic reduction of the ketopantoic acid to d-(-)-pantoic acid. After the reaction d-(-)-pantoic acid could be lactonized by means of acid treatment. During the conversion, the d-(-)-isomer, which was initially present in the reaction mixture, did not undergo any modification.     

Ketopantoic acid reductase of Pseudomonas maltophilia 845. Purification, characterization, and role in pantothenate biosynthesis

Ketopantoic acid reductase (EC 1.1.1.169), an enzyme that catalyzes the formation of D-(-)-pantoic acid from ketopantoic acid, was purified 6,000-fold to apparent homogeneity with a 35% overall recovery from Pseudomonas maltophilia 845 and then crystallized. The relative molecular mass of the native enzyme, as estimated by the sedimentation equilibrium method, is 87,000 +/- 5,000, and the subunit molecular mass is 30,500. The enzyme shows high specificity for ketopantoic acid as a substrate (Km = 400 microM, Vm = 1,310 units/mg of protein) and NADPH as a coenzyme (Km = 31.8 microM). Only 2-keto-3-hydroxyisovalerate (Km = 8.55 mM, Vm = 35.8 units/mg) was reduced among a variety of other carbonyl compounds tested. The reaction is reversible (Km for D-(-)-pantoic acid = 52.1 mM), although the reaction equilibrium greatly favors the direction of D-(-)-pantoic acid formation. That the enzyme is responsible for the synthesis of D-(-)-pantoic acid necessary for the biosynthesis of pantothenic acid in P. maltophilia 845 is indicated by the observations that only this enzyme is missing in D-(-)-pantoate (or pantothenate)-requiring mutants derived from P. maltophilia 845 among several enzymes (i.e. ketopantoyl lactone reductase (EC 1.1.1.168) and acetohydroxy acid isomeroreductase (EC 1.1.1.86], which may be concerned in the formation of D-(-)-pantoic acid, assayed, whereas it is present in substantial amounts in the parent strain and in spontaneous revertants of the mutants.     

A novel fungal enzyme, NADPH-dependent carbonyl reductase, showing high specificity to conjugated polyketones. Purification and characterization

A novel enzyme which specifically catalyzes the reduction of conjugated polyketones was purified to homogeneity from cells of Mucor ambiguus AKU 3006. The enzyme has a strict requirement for NADPH and irreversibly reduces a number of quinones such as p-benzoquinone, alpha-naphthoquinone and acenaphthenequione. The enzyme also reduces polyketones such as isatin and ketopantoyl lactone, and their derivatives. The apparent Km values for isatin and ketopantoyl lactone are 49.9 microM and 714 microM, respectively. The reduction of ketopantoyl lactone proceeds stereospecifically to yield L-(+)-pantoyl lactone. The pro-S (A) hydrogen at C-4 of NADPH is transferred to the substrate. The enzyme is not a flavoprotein and consists of two polypeptide chains with an identical relative molecular mass of 27,500. Quercetin, dicoumarol and some SH reagents inhibit the enzyme activity. 3-Methyl-1,2-cyclopentanedione and 1,3-cyclohexanedione are uncompetitive inhibitors with Ki values of 80.9 microM and 64.5 microM, respectively, to ketopantoyl lactone.     

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to d-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2′-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.     

Enzymatic in situ determination of stereospecificity of NAD-dependent dehydrogenases

Amino acid racemases inherently catalyze the exchange of alpha-hydrogen of amino acids with deuterium during racemization in 2H2O. When the reactions catalyzed by alanine racemase (EC 5.1.1.1) and L-alanine dehydrogenase (EC 1.4.1.1), which is pro-R specific for the C-4 hydrogen transfer of NADH, are coupled in 2H2O, [4R-2H]NADH is exclusively produced. Similarly, [4S-2H]NADH is made in 2H2O with amino-acid racemase with low substrate specificity (EC 5.1.1.10) and L-leucine dehydrogenase (EC 1.4.1.9), which is pro-S specific. We have established a simple procedure for the in situ analysis of stereospecificity of C-4 hydrogen transfer of NADH by an NAD-dependent dehydrogenase by combination with either of the above two couples of enzymes in the same reaction mixture. When the C-4 hydrogen of NAD+ is fully retained after sufficient incubation, the stereospecificity of hydrogen transfer by a dehydrogenase is the same as that of alanine dehydrogenase or leucine dehydrogenase. However, when the C-4 hydrogen of NAD+ is exchanged with deuterium, the enzyme to be examined shows the different stereospecificity from alanine dehydrogenase or leucine dehydrogenase. Thus, we can readily determine the stereospecificity by 1H NMR measurement without isolation of the coenzymes and products.     

The stereospecificity of the reduction of nitrate by reduced nicotinamide-adenine dinucleotides catalysed by Candida utilis preparations.

The reduction of nitrate by reduced nicotinamide-adenine dinucleotides, catalysed by extract of Candida utilis, exhibits an apparent high degree of stereospecificity for the 'B' methylene hydrogen atom of NADPH and mixed stereospecificity for the methylene hydrogen atoms of NADH. Purified nitrate reductase, on the other hand, exhibits 'A' stereospecificity for NADH and NADPH. The apparent switch of stereospecificity from the 'B' to the 'A' side of NADPH, which occurs after purification of the enzyme, is partly explained by the fact that in crude extracts nitrate is reduced completely to ammonia. Nitrite does not accumulate but is reduced to ammonia by nitrite dehydrogenase, which is 'B'-specific, so that up to 75% of hydrogen removed from NADPH during the reduction of nitrate could occur from the 'B' side. A further increase in the removal of hydrogen from the 'B' side of NADPH could be the kinetic isotope effect that is observed when ['A'-3H]NADPH is the reductant, the H--C bond being cleaved 2.3 times faster than the 3H--C bond. The mixed stereospecificity observed with NADH has been traced to an uncharacterized enzyme that catalyses a 'B'-specific exchange between NAD+ and NADH. This reaction is discussed in relation to the possibility that it may explain other cases of apparent mixed stereospecificity that have been reported.     

Glutathione reductase: comparison of steady-state and rapid reaction primary kinetic isotope effects exhibited by the yeast, spinach, and Escherichia coli enzymes

Kinetic parameters for NADPH and NADH have been determined at pH 8.1 for spinach, yeast, and E. coli glutathione reductases. NADPH exhibited low Km values for all enzymes (3-6 microM), while the Km values for NADH were 100 times higher (approximately 400 microM). Under our experimental conditions, the percentage of maximal velocities with NADH versus those measured with NADPH were 18.4, 3.7, and 0.13% for the spinach, yeast, and E. coli enzymes, respectively. Primary deuterium kinetic isotope effects were independent of GSSG concentration between Km and 15Km levels, supporting a ping-pong kinetic mechanism. For each of the three enzymes, NADPH yielded primary deuterium kinetic isotope effects on Vmax only, while NADH exhibited primary deuterium kinetic isotope effects on both V and V/K. The magnitude of DV/KNADH at pH 8.1 is 4.3 for the spinach enzyme, 2.7 for the yeast enzyme, and 1.6 for the E. coli glutathione reductase. The experimentally determined values of TV/KNADH of 7.4, 4.2, and 2.2 for the spinach, yeast, and E. coli glutathione reductases agree well with those calculated from the corresponding DV/KNADH using the Swain-Schaad expression. This suggests that the intrinsic primary kinetic isotope effect on NADH oxidation is fully expressed. In order to confirm this conclusion, single-turnover experiments have been performed. The measured primary deuterium kinetic isotope effects on the enzyme reduction half-reaction using NADH match those measured in the steady state for each of the three glutathione reductases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereospecificity of hydrogen transfer by bovine testicular 20 alpha-hydroxysteroid dehydrogenase

The stereospecificity of hydrogen transfer between steroid (17-hydroxyprogesterone) and both natural cofactors by bovine testicular 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) has been determined. Cofactors used in these studies, [4-pro-S-3H]NADH ([4B-3H]NADH) and [4-pro-S-3H]NADPH ([4B-3H]NADPH) were generated with human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) utilizing [17 alpha-3H]estradiol-17 beta and NAD+ or NADP+, respectively. The resulting [4B-3H]NADH and [4B-3H]NADPH were purified by ion-exchange chromatography and separately incubated with molar excess of 17-hydroxyprogesterone as substrate in the presence of 20 alpha-HSD. Following incubation, steroid reactant and product were extracted, separated by HPLC and quantitated as to mass and content of tritium. The oxidized and reduced cofactors were separated by ion-exchange chromatography and quantitated as to mass and tritium content. In all incubations, equimolar amounts of 17,20 alpha-dihydroxy-4-pregnen-3-one and oxidized cofactor were obtained. Further, all recovered radioactivity remained with cofactor and none was found in the steroid product. In additional experiments, both reduced cofactors were separately incubated with glutamate dehydrogenase, an enzyme known to transfer from the B-side of the nicotinamide ring. Here radioactivity was present only in the unreacted cofactor fractions and in the product, glutamic acid. The results indicate that bovine testicular 20 alpha-HSD catalyzes transfer of the 4A-hydrogen from the dihydronicotinamide moiety of the reduced cofactor. Finally, this work described modifications that represent considerable improvement in the purification and assay of bovine 20 alpha-HSD as originally described.     

Engineered glycolytic glyceraldehyde-3-phosphate dehydrogenase binds the anti conformation of NAD+ nicotinamide but does not experience A-specific hydride transfer

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a NAD-dependent oxidoreductase which catalyzes the oxidative phosphorylation of d-glyceraldehyde-3-phosphate (G3P) to form 1, 3-diphosphoglycerate. The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation. GAPDH is classified as a B-specific oxidoreductase. The inspection of several crystal structures of GAPDHs indicates that the efficient hydride transfer from the hemithioacetal intermediate to the C4 position of the pyridinium si face requires optimal nicotinamidium-protein contacts for a suitable pyridinium-ring orientation. In previous studies carried out on Escherichia coli GAPDH (C. Corbier, A. Mougin, Y. Mely, H. W. Adolph, M. Zeppezauer, D. Gerard, A. Wonacott, and G. Branlant, Biochimie 72, 545-554, 1990; J. Eyschen, C. Corbier, B. Vitoux, G. Branlant, and M. T. Cung, Protein Pept. Lett. 1, 19-24, 1994), the role of the invariant Asn 313 residue, as an anchor which favors the syn orientation of the nicotinamide ring, was examined. Here, we report further investigations on the molecular factors responsible for the cofactor stereospecificity. Two single [Gly317] and [Ala317] GAPDH mutants and one double [Thr313-Gly317] GAPDH mutant were constructed on the basis of a molecular modelling study from the crystal structure of holo GAPDH from E. coli (E. Duée, L. Olivier-Deyris, E. Fanchon, C. Corbier, G. Branlant, and O. Dideberg, J. Mol. Biol. 257, 814-838, 1996). The Kd constants of [Ala317], [Gly317], and [Thr313-Gly317] GAPDH mutants for NAD are 5, 13, and 300 times higher than that of wild-type GAPDH. Transferred nuclear Overhauser effect spectroscopy demonstrates that the wild-type syn orientation of bound nicotinamide remains unchanged in the [Gly317] and [Ala317] mutants, whereas a conformational equilibrium between the syn and anti forms occurs in the [Thr313-Gly317] double mutant with a preference for the anti conformer. Although the double mutant preferably binds the nicotinamide ring in an anti conformation, it still exhibits B hydride transfer stereospecificity. Yet, the catalytic efficiency is much less than that of the wild type. This indicates that the holo GAPDH mutant fraction with an anti orientation of bound NAD is not capable of forming the ternary complex with G3P which would be required for an efficient A-specific catalytic process. The reasons of this catalytic inefficiency are discussed in relation with the historical and functional models which were advanced to explain the stereospecificity of NAD(P)-dependent dehydrogenases.     

Stereochemistry of the reactions catalyzed by chicken liver fatty acid synthase

The stereochemistry of the four partial reactions catalyzed by chicken liver fatty acid synthase that lead to the synthesis of palmitic acid has been determined. The reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA by NADPH proceeds with the transfer of the pro-4S hydrogen of NADPH to form D-3-hydroxybutyryl-CoA. During the subsequent dehydration of D-3-hydroxybutyryl-CoA the pro-2S hydrogen and the 3-hydroxyl group are removed in a syn elimination to form crotonyl-CoA. Crotonyl-CoA is reduced to butyryl-CoA by NADPH, with the transfer of the pro-4R hydrogen of NADPH to the pro-3R position in butyryl-CoA and the transfer of a solvent hydrogen to the pro-2S position. The occurrence of the syn dehydration, when combined with the results of a previous study [ Sedgwick , B., & Cornforth , J. W. (1977) Eur. J. Biochem. 75, 465-479], implies that the condensation of the enzyme-bound malonyl moiety with the enzyme-bound saturated fatty acid to form a 3-keto intermediate proceeds with inversion at C-2 of the malonyl. The stereochemistry of the hydration was derived from an analysis of the spin-spin coupling constant of 3-hydroxy[2-2H]butyric acid benzylamides obtained from 3-hydroxy[2-2H]butyryl-CoA synthesized by fatty acid synthase. The elucidation of the stereochemistry of the reduction of crotonyl-CoA relied on the previously established stereochemistry of pork liver acyl-CoA dehydrogenase. The source of all 28 prochiral hydrogens of the palmitic acid synthesized by chicken liver fatty acid synthase was inferred from the results of this work.     

Glutathione reductases from a variety of sources are inhibited by physiological levels of glutathione.

1. Glutathione reductase from human platelets, bovine intestinal mucosa, yeast and E. coli were inhibited in vitro by physiological levels of reduced glutathione with IC50s of 6.61 mM, 2.92 mM, 2.40 mM and 12.11 mM, respectively. 2. A steady-state kinetic examination revealed that glutathione inhibited the NADPH oxidation (at constant [glutathione-disulphide]) catalysed by the eucaryotic enzymes uncompetitively, whereas the E. coli enzyme appeared unaffected by glutathione concentrations of up to 10 mM. 3. With respect to glutathione inhibition of glutathione-disulphide reduction (at constant [NADPH]), the human enzyme was inhibited uncompetitively; the bovine and yeast enzymes displayed apparent mixed hyperbolic inhibition; the E. coli enzyme was inhibited competitively.     

The stereospecificities of seven dehydrogenases from Acholeplasma laidlawii. The simplest historical model that explains dehydrogenase stereospecificity

Stereospecificities are reported for seven dehydrogenases from Acholeplasma laidlawii, an organism from an evolutionarily distinct branch of life which has not previously been studied from a stereochemical point of view. Three of the activities examined (alcohol dehydrogenase, lactate dehydrogenase, and alanine dehydrogenase) catalyze the transfer of the pro-R (A) hydrogen from NADH. Four other activities (3-hydroxy-3-methylglutaryl-CoA reductase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, and NADH oxidase) catalyze the transfer of the pro-S (B) hydrogen from NAD(P)H. The stereospecificity of hydroxymethylglutaryl-CoA reductase is notable because it is the opposite of that of hydroxymethylglutaryl-CoA reductases from yeast and rat. These data are used to derive the simplest historical model capable of explaining available experimental facts.     

Characterization and stereochemistry of cofactor oxidation by a type II dihydrofolate reductase

Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are different both in sequence and in structure from known chromosomal DHFRs. These plasmid-derived DHFRs are responsible for conferring trimethoprim resistance to the host strain. A derivative of R388 DHFR, RBG200, has been cloned and overproduced [Vermersch, P. S., Klass, M. R., & Bennett, G. N. (1986) Gene 41, 289]. With this cloned and overproduced protein, a rapid purification procedure has been developed that yields milligram quantities of apparently homogeneous RBG200 DHFR with a specific activity 1.5-fold greater than that previously reported for the purified R388 protein [Amyes, S. G. B., & Smith, J. T. (1976) Eur. J. Biochem. 61, 597]. The pH versus activity profile and the native molecular weight of RBG200 DHFR were found to be similar to those previously reported for other type II DHFRs but different from those of the known chromosomal DHFRs. Stereospecifically labeled [4(S)-2H,4(R)-1H]NADPH was synthesized and used to determine the stereospecificity of NADPH oxidation by RBG200 DHFR. RBG200 DHFR was found to specifically transfer the pro-R hydrogen of NADPH to dihydrofolate, making it a member of the A-stereospecific class of dehydrogenases. Thus, although RBG200 DHFR is different both in sequence and in structure from known chromosomal enzymes, both enzymes catalyze identical hydrogen-transfer reactions. Two distinct binary RBG200 DHFR-NADP+ complexes were detected by monitoring the 1H NMR chemical shifts and line widths of the coenzyme in the presence of RBG200 DHFR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene cloning and overexpression of two conjugated polyketone reductases,novel aldo-keto reductase family enzymes,of<Emphasis Type="Italic"> Candida parapsilosis</Emphasis>

The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to d-pantoyl lactone.     

l-Pantoyl lactone dehydrogenase from Rhodococcus erythropolis: genetic analyses and application to the stereospecific oxidation of l-pantoyl lactone

The 1,2-propanediol (1,2-PD) inducible membrane-bound L-pantoyl lactone (L-PL) dehydrogenase (LPLDH) has been isolated from Rhodococcus erythropolis AKU2103 (Kataoka et al. in Eur J Biochem 204:799, 1992). Based on the N-terminal amino acid sequence of LPLDH and the highly conserved amino acid sequence in homology search results, the LPLDH gene (lpldh) was cloned. The gene consists of 1,179 bases and encodes a protein of 392 amino acid residues. The deduced amino acid sequence showed high similarity to the proteins of the FMN-dependent α-hydroxy acid dehydrogenase/oxidase family. The overexpression vector pKLPLDH containing lpldh with its upstream region (1,940 bp) was constructed and introduced into R. erythropolis AKU2103. The recombinant R. erythropolis AKU2103 harboring pKLPLDH showed six times higher LPLDH activity than the wild-type strain. Conversion of L-PL to ketopantoyl lactone was achieved with 92% or 80% conversion yield when the substrate concentration was 0.768 or 1.15 M, respectively. Stereoinversion of L-PL to D-PL was also carried out by using the combination of recombinant R. erythropolis AKU2103 harboring pKLPLDH and ketopantoic acid-reducing Escherichia coli.     

Enzymatic synthesis of [4R-2H]NAD (P)H and [4S-2H]NAD(P)H and determination of the stereospecificity of 7 alpha- and 12 alpha hydroxysteroid dehydrogenase

The stereospecifically labeled coenzymes [4R-2H]NADH, [4R-2H]NADPH and [4S-2H]NAD(P)H were synthesized enzymatically in high yield and high isotopic purity (greater than or equal to 95%) with 2HCOO2H/formate dehydrogenase, (CH3)2C2HOH/alchol dehydrogenase from Thermoanaerobium brockii and [1-2H]glucose/glucose dehydrogenase, respectively. This set of deuterated coenzymes was used to determine the stereospecificity of the previously unstudied 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli (NAD-dependent) and 12 alpha-hydroxysteroid dehydrogenase from Clostridium group P (NADP-dependent). H-NMR and EI-MS of the nicotinamide moiety after enzymatic oxidation of deuterated NAD(P)H with dehydrocholic acid as substrate showed that both dehydrogenases are B-sterospecific.     

Studies on the metabolism of unsaturated fatty acids. XII. Reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli

Incorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli (E. coli) was investigated. When trans-2,cis-4-decadienoyl-CoA was incubated with 4R- or 4S-[4-2H1]NADPH in the presence of purified 2,4-dienoyl-CoA reductase, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to its pyrrolidine amide. On the other hand, when the dienoyl-CoA was incubated in the presence of NADPH and the reductase in 2H2O, two deuterium atoms were incorporated: One deuterium atom was located at the C-4 position of trans-2-decenoate, and the other at the C-5 position. The UV and shorter wavelengths of the visible spectrum of the reductase solution revealed that the reductase contained flavin as a prosthetic group. Therefore it is considered that a hydrogen atom of NADPH was first transferred to the flavin moiety of the reductase, and then the hydrogen atom was rapidly exchanged for one in the medium before its direct transfer to the substrate.     

Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase.

Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9'-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9'-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.