Identification of the substrate radical intermediate derived from ethanolamine during catalysis by ethanolamine ammonia-lyase

Rapid-mix freeze-quench (RMFQ) methods and electron paramagnetic resonance (EPR) spectroscopy have been used to characterize the steady-state radical in the deamination of ethanolamine catalyzed by adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL). EPR spectra of the radical intermediates formed with the substrates, [1-13C]ethanolamine, [2-13C]ethanolamine, and unlabeled ethanolamine were acquired using RMFQ trapping methods from 10 ms to completion of the reaction. Resolved 13C hyperfine splitting in EPR spectra of samples prepared with [1-13C]ethanolamine and the absence of such splitting in spectra of samples prepared with [2-13C]ethanolamine show that the unpaired electron is localized on C1 (the carbinol carbon) of the substrate. The 13C splitting from C1 persists from 10 ms throughout the time course of substrate turnover, and there was no evidence of a detectable amount of a product like radical having unpaired spin on C2. These results correct an earlier assignment for this radical intermediate [Warncke, K., et al. (1999) J. Am. Chem. Soc. 121, 10522-10528]. The EPR signals of the substrate radical intermediate are altered by electron spin coupling to the other paramagnetic species, cob(II)alamin, in the active site. The dipole-dipole and exchange interactions as well as the 1-13C hyperfine splitting tensor were analyzed via spectral simulations. The sign of the isotropic exchange interaction indicates a weak ferromagnetic coupling of the two unpaired electrons. A Co2+-radical distance of 8.7 A was obtained from the magnitude of the dipole-dipole interaction. The orientation of the principal axes of the 13C hyperfine splitting tensor shows that the long axis of the spin-bearing p orbital on C1 of the substrate radical makes an angle of approximately 98 degrees with the unique axis of the d(z2) orbital of Co2+.     

Analysis of the electron paramagnetic resonance spectrum of a radical intermediate in the coenzyme B(12)-dependent ethanolamine ammonia-lyase catalyzed reaction of S-2-aminopropanol

The structure of the steady-state radical intermediate in the deamination of S-2-aminopropanol catalyzed by ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been probed by electron paramagnetic resonance (EPR) spectroscopy using isotopically labeled forms of the substrate and of the adenosylcobalamin cofactor. Electron spin-spin coupling between the radical, centered on the carbon skeleton of the substrate, and the low-spin Co(2+) in cob(II)alamin (B(12r)) produces a dominant splitting of the EPR signals of both the radical and the Co(2+). Analysis of the exchange and dipole-dipole contributions to the spin-spin coupling indicates that the two paramagnetic centers are separated by approximately 11 A. Experiments with (13)C- and with (2)H-labeled forms of S-2-aminopropanol show that the radical is centered on C1 of the carbon skeleton of the substrate in agreement with an earlier report [Babior, B. M., Moss, T. H., Orme-Johnson, W. H., and Beinert, H., (1974) J. Biol. Chem. 249, 4537-4544]. Experiments with perdeutero-S-2-aminopropanol and [2-(15)N]-perdeutero-S-2-aminopropanol reveal a strong hyperfine splitting from the substrate nitrogen, which indicates that the radical is the initial substrate radical created by abstraction of a hydrogen atom from C1 of S-2-aminopropanol. The strong nitrogen hyperfine splitting further indicates that the amino substituent at C2 is approximately eclipsed with respect to the half-occupied p orbital at C1. Experiments with adenosylcobalamin enriched in (15)N in the dimethylbenzimidazole moiety show that the axial base of the cofactor remains attached to the Co(2+) in a functional steady-state reaction intermediate.     

Hydrazine cation radical in the active site of ethanolamine ammonia-lyase: mechanism-based inactivation by hydroxyethylhydrazine.

A study has been made of the mechanism of inactivation of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase (EAL), by hydroxyethylhydrazine. Incubation of EAL with adenosylcobalamin and hydroxyethylhydrazine, an analogue of ethanolamine, leads to rapid and complete loss of enzymic activity. Equimolar quantities of 5'-deoxyadenosine, cob(II)alamin (B(12r)), hydrazine cation radical, and acetaldehyde are products of the inactivation. Inactivation is attributed to the tight binding of B(12r) in the active site. Removal of B(12r) from the protein by ammonium sulfate precipitation under acidic conditions, however, restores significant activity. This inactivation event has also been monitored by electron paramagnetic resonance (EPR) spectroscopy. In addition to EPR signals associated with B(12r), spectra of samples of inactivation mixtures reveal the presence of another radical. The other radical is bound in the active site where it undergoes weak magnetic interactions with the low spin Co(2+) in B(12r). The radical species was unambiguously identified as a hydrazine cation radical by using [(15)N(2)]hydroxyethylhydrazine, (2)H(2)O, and quantitative interpretation of the EPR spectra. Homolytic fragmentation of a hydroxyethylhydrazine radical to acetaldehyde and a hydrazine cation radical is consistent with all of the observations. All of the experiments indicate that the mechanism-based inactivation of EAL by hydroxyethylhydrazine results from irreversible cleavage of the cofactor and tight binding of B(12r) to the active site.     

Revisiting the bilayer structures of fluid phase phosphatidylglycerol lipids: Accounting for exchangeable hydrogens

We recently published two papers detailing the structures of fluid phase phosphatidylglycerol (PG) lipid bilayers (Ku?erka et al., 2012 J. Phys. Chem. B 116: 232–239; Pan et al., 2012 Biochim. Biophys. Acta Biomembr. 1818: 2135–2148), which were determined using the scattering density profile model. This hybrid experimental/computational technique utilizes molecular dynamics simulations to parse a lipid bilayer into components whose volume probabilities follow simple analytical functional forms. Given the appropriate scattering densities, these volume probabilities are then translated into neutron scattering length density (NSLD) and electron density (ED) profiles, which are used to jointly refine experimentally obtained small angle neutron and X-ray scattering data. However, accurate NSLD and ED profiles can only be obtained if the bilayer's chemical composition is known. Specifically, in the case of neutron scattering, the lipid's exchangeable hydrogens with aqueous D₂O must be accounted for, as they can have a measureable effect on the resultant lipid bilayer structures. This was not done in our above-mentioned papers. Here we report on the molecular structures of PG lipid bilayers by appropriately taking into account the exchangeable hydrogens. Analysis indicates that the temperature-averaged PG lipid areas decrease by 1.5 to 3.8 Å², depending on the lipid's acyl chain length and unsaturation, compared to PG areas when hydrogen exchange was not taken into account.     

Formation of transient complexes in the glutamate dehydrogenase catalyzed reaction.

The reaction of glutamate dehydrogenase and glutamate (gl) with NAD+ and NADP+ has been studied with stopped-flow techniques. The enzyme was in all experiments present in excess of the coenzyme. The results indicate that the ternary complex (E-NAD(P)H-kg) is present as an intermediate in the formation of the stable complex (E-NAD(P)H-gl). The identification of the complexes is based on their absorption spectra. The binding of the coenzyme to (E-gl) is the rate-limiting step in the formation of (E-NAD(P)H-kg) while the dissociation of alpha-ketoglutarate (kg) from this complex is the rate-limiting step in the formation of (E-NAD(P)H-gl). The Km for glutamate was 20-25 mM in the first reaction and 3 mM in the formation of the stable complex. The Km values were independent of the coenzyme. The reaction rates with NAD+ were approximately 50% greater than those with NADP+. Furthermore, high glutamate concentration inhibited the formation of (E-NADH-kg) while no substrate inhibition was found with NADP+ as coenzyme. ADP enhanced while GTP reduced the rate of (E-NAD(P)H-gl) formation. The rate of formation of (E-NAD(P)H-kg) was inhibited by ADP, while it increased at high glutamate concentration when small amounts of GTP were added. The results show that the higher activity found with NAD+ compared to NADP+ under steady-state assay conditions do not necessarily involve binding of NAD+ to the ADP activating site of the enzyme. Moreover, the substrate inhibition found at high glutamate concentration under steady-state assay condition is not due to the formation of (E-NAD(P)H-gl) as this complex is formed with Km of 3 mM glutamate, and the substrate inhibition is only significant at 20-30 times this concentration.     

Entropy effects on protein hinges: the reaction catalyzed by triosephosphate isomerase

Many proteins utilize segmental motions to catalyze a specific reaction. The Omega loop of triosephosphate isomerase (TIM) is important for preventing the loss of the reactive enediol(ate) intermediate. The loop opens and closes even in the absence of the ligand, and the loop itself does not change conformation during movement. The conformational changes are localized to two hinges at the loop termini. Glycine is never observed in native TIM hinge sequences. In this paper, the hypothesis that limited access to conformational space is a requirement for protein hinges involved in catalysis was tested. The N-terminal hinge was mutated to P166/V167G/W168G (PGG), and the C-terminal hinge was mutated to K174G/T175G/A176G (GGG) in chicken TIM. The single-hinge mutants PGG and GGG had k(cat) values 200-fold lower than that of the wild type and K(m) values 10-fold higher. The k(cat) of double-hinge mutant P166/V167G/W168G/K174G/T175G/A176G was reduced 2500-fold; the K(m) was 10-fold higher. A combination of primary kinetic isotope effect measurements, isothermal calorimetric measurements, and (31)P NMR spectroscopic titration with the inhibitor 2-phosphoglycolate revealed that the mutants have a different ligand-binding mode than that of the wild-type enzyme. The predominant conformations of the mutants even in the presence of the inhibitor are loop-open conformations. In conclusion, mutation of the hinge residues to glycine resulted in the sampling of many more hinge conformations with the consequence that the population of the active-closed conformation is reduced. This reduced population results in a reduced catalytic activity.     

Solvent isotope effects on the reaction catalyzed by yeast hexokinase

The pH dependence of the maximum velocity (V) for the phosphorylation of glucose, the V/Kglucose and the V/KMgATP have been obtained in H2O and 2H2O. In H2O, V decreases below a pK of 5.8, V/Kglucose decreases below a pK of 6.1 and V/KMgATP decreases below a pK of 6.7. In 2H2O, complex behavior is observed for these parameters as a function of pD. The ratios of the parameters in H2O and 2H2O above their respective pK values give solvent deuterium isotope effects of about 1.5-1.7 for all three parameters. When 1,5-anhydromannitol is used as an alternative substrate, an isotope effect different than unity is obtained only for V/K1,5-anhydromannitol which gives a value of about 0.7. Both the complex pH profiles and the relative magnitude of the isotope effects are interpreted in terms of a pH-dependent change in the E X glucose complex.     

Isotope and elemental effects indicate a rate-limiting methyl transfer as the initial step in the reaction catalyzed by Escherichia coli cyclopropane fatty acid synthase

Cyclopropane fatty acid (CFA) synthases catalyze the formation of cyclopropane rings on unsaturated fatty acids (UFAs) that are natural components of membrane phospholipids. The methylene carbon of the cyclopropane ring derives from the activated methyl group of S-adenosyl-L-methionine (AdoMet), affording S-adenosyl-L-homocysteine (AdoHcys) and a proton as the remaining products. This reaction is unique among AdoMet-dependent enzymes, because the olefin of the UFA substrate is isolated and unactivated toward nucleophilic or electrophilic addition, raising the question as to the timing and mechanism of proton loss from the activated methyl group of AdoMet. Two distinct reaction schemes have been proposed for this transformation; however, neither was based on detailed in vitro mechanistic analysis of the enzyme. In the preceding paper [Iwig, D. F. and Booker, S. J. (2004) Biochemistry 43, http://dx.doi.org/10.1021/bi048693+], we described the synthesis of two analogues of AdoMet, Se-adenosyl-L-selenomethionine (SeAdoMet) and Te-adenosyl-L-telluromethionine (TeAdoMet), and their intrinsic reactivity toward polar chemistry in which AdoMet is known to be involved. We found that the electrophilicity of AdoMet and its onium congeners followed the series SeAdoMet > AdoMet > TeAdoMet, while the acidity of the carbons adjacent to the relevant heteroatom followed the series AdoMet > SeAdoMet > TeAdoMet. When each of these compounds was used as the methylene donor in the CFA synthase reaction, the kinetic parameters of the reaction, k(cat) and k(cat) K(M)(-1), followed the series SeAdoMet > AdoMet > TeAdoMet, suggesting that the reaction takes place via methyl transfer followed by proton loss, rather than by processes that are initiated by proton abstraction from AdoMet. Use of S-adenosyl-L-[methyl-d(3)]methionine as the methylene donor resulted in an inverse isotope effect of 0.87 +/- 0.083, supporting this conclusion and also indicating that the methyl transfer takes place via a tight s(N)2 transition state.     

A transient intermediate in the reaction catalyzed by (S)-mandelate dehydrogenase from Pseudomonas putida

(S)-Mandelate dehydrogenase from Pseudomonas putida is a member of a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids to alpha-ketoacids. The reductive half-reaction consists of the steps involved in substrate oxidation and FMN reduction. In this study, we investigated the mechanism of this half-reaction in detail. At low temperatures, a transient intermediate was formed in the course of the FMN reduction reaction. This intermediate is characteristic of a charge-transfer complex of oxidized FMN and an electron-rich donor and is formed prior to full reduction of the flavin. The intermediate was not due to binding of anionic substrates or inhibitors. It was only observed with efficient substrates that have high k(cat) values. At higher temperatures, it was formed within the dead time of the stopped-flow instrument. The rate of formation of the intermediate was 3-4-fold faster than its rate of disappearance; the former had a larger isotope effect. This suggests that the charge-transfer donor is an electron-rich carbanion/enolate intermediate that is generated by the base-catalyzed abstraction of the substrate alpha-proton. This is consistent with the observation that the intermediate was not observed with the R277K and R277G mutants, which have been shown to destabilize the carbanion intermediate (Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065). Thus, the MDH reaction has two rate-limiting steps of similar activation energies: the formation and breakdown of a distinct intermediate, with the latter step being slightly more rate limiting. We also show that MDH is capable of catalyzing the reverse reaction, the reoxidation of reduced MDH by the product ketoacid, benzoylformate. The transient intermediate was observed during the reverse reaction as well, confirming that it is indeed a true intermediate in the MDH reaction pathway.     

Kinetics of the transhydrogenase reaction catalyzed by mitochondrial NADH:ubiquinone oxidoreductase (complex I)

The kinetics of the NADH3'-acetylpyridine adenine dinucleotide (APAD⁺) transhydrogenase reaction (DD-reaction) catalyzed by different preparations of mitochondrial NADH-dehydrogenase (submitochondrial particles (SMP), purified Complex I, and three-subunit fragment of Complex I (FP)) have been studied. Complex I (in SMP or in purified preparation) catalyzes two NADHAPAD⁺ reactions with different rates and nucleotide affinities. Reaction 1 has high affinity to APAD⁺ (K _m = 7 M, for SMP) and low rate (V _m = 0.2 mol/min per mg protein, for SMP) and occurs with formation of a ternary complex. Reaction 2 has much higher rate and considerably lower affinity for oxidized nucleotide (V _m = 1.7 mol/min per mg protein and K _m = 160 M, for SMP). FP catalyzes only reaction 1. ADP-ribose inhibits reaction 1 with mixed type inhibition (competitive with non-competitive) with respect to NADH and APAD⁺. Rhein competes with both substrates. The results suggest that at least two nucleotide-binding sites exist in Complex I.     

The role of the charge transfer complex in the transketolase catalyzed reaction

Thiamine pyrophosphate (TPP), when bound with transketolase (TK) induces some changes in the absorption of the enzyme and coenzyme which can be registered by difference spectrophotometry. The binding of a donor substrate to the binary complex give rise to changes in the absorption region of the TPP thiazolium ring and in the charge transfer spectrum. With low concentrations of hydroxypyruvate, the kinetics of these changes may be revealed. The possibility is discussed of the charge transfer complex (CTC) being involved in the catalytic reaction.     

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Reaction of the enzyme.cofactor complex with 2-aminoacetaldehyde

Ethanolamine ammonia-lyase (EC 4.3.1.7) catalyzes the adenosylcobalamin-dependent deamination of ethanolamine and 2-aminopropanol. Incubation of the enzyme.cofactor complex with 2-aminoacetaldehyde leads to rapid cleavage of the carbon--cobalt bond accompanied by the destruction of the corrinoid portion of the cofactor. During this reaction the adenosyl portion of the cofactor is oxidized to 4',5'-anhydroadenosine, and the aminoacetaldehyde is converted to acetic acid, which remains associated with the enzyme as a noncovalent complex which survives gel filtration. There is no evidence for the alkylation of the corrin metal by the substrate analog. The enzyme.AdoCbl complex is thus able to eliminate an amino group from a substrate analog without the formation of a new alkyl cobalamin in which the analog is a ligand. These observations do not support the participation of what might be termed "substratylcobalamin" as an intermediate in the ammonia migration occurring in reactions catalyzed by ethanolamine ammonia-lyase.