The effects of monoamine oxidase B inhibition on dopamine metabolism in rats with nigro-striatal lesions

The purpose of this study was to examine whether monoamine oxidase type B (MAO-B) has a role in striatal dopamine metabolism in animals with a unilateral lesion of the medial forebrain bundle, and whether 2-phenylethylamine (PE) could have a role in amplification of dopamine (DA) responses in DA depleted striatum. Inhibition of MAO-B did not alter DA metabolism in lesioned striata. PE accumulation decreased with loss of DA as long as there was no DA dysfunction. In lesioned striata with dysfunction of DA transmission at the synaptic level, PE accumulation increased,suggesting a compensatory increase in PE synthesis. This increase in PE levels does not appear to be mediated by an increase in the total striatal aromaticl-amino acid decarboxylase (AADC) activity. We conclude that inhibition of MAO-B has no effect on DA metabolism in the hemi-parkinsonian rat striatum and that PE could be involved in the antiparkinsonian action of MAO-B inhibitors.     

Molecular mechanism of intramolecular electron transfer in dimeric sulfite oxidase

Sulfite oxidase (SOX) is a homodimeric molybdoheme enzyme that oxidizes sulfite to sulfate at the molybdenum center. Following substrate oxidation, molybdenum is reduced and subsequently regenerated by two sequential electron transfers (ETs) via heme to cytochrome c. SOX harbors both metals in spatially separated domains within each subunit, suggesting that domain movement is necessary to allow intramolecular ET. To address whether one subunit in a SOX dimer is sufficient for catalysis, we produced heterodimeric SOX variants with abolished sulfite oxidation by replacing the molybdenum-coordinating and essential cysteine in the active site. To further elucidate whether electrons can bifurcate between subunits, we truncated one or both subunits by deleting the heme domain. We generated three SOX heterodimers: (i) SOX/Mo with two active molybdenum centers but one deleted heme domain, (ii) SOX/Mo_C264S with one unmodified and one inactive subunit, and (iii) SOX_C264S/Mo harboring a functional molybdenum center on one subunit and a heme domain on the other subunit. Steady-state kinetics showed 50% SOX activity for the SOX/Mo and SOX/Mo_C264S heterodimers, whereas SOX_C264S/Mo activity was reduced by two orders of magnitude. Rapid reaction kinetics monitoring revealed comparable ET rates in SOX/Mo, SOX/Mo_C264S, and SOX/SOX, whereas in SOX_C264S/Mo, ET was strongly compromised. We also combined a functional SOX Mo domain with an inactive full-length SOX R217W variant and demonstrated interdimer ET that resembled SOX_C264S/Mo activity. Collectively, our results indicate that one functional subunit in SOX is sufficient for catalysis and that electrons derived from either Mo^(IV) or Mo^(V) follow this path.     

A lysine conserved in the monoamine oxidase family is involved in oxidation of the reduced flavin in mouse polyamine oxidase

Lysine 315 of mouse polyamine amine oxidase corresponds to a lysine residue that is conserved in the flavoprotein amine oxidases of the monoamine oxidase structural family. In several structures, this lysine residue forms a hydrogen bond to a water molecule that is hydrogen-bonded to the flavin N(5). Mutation of Lys315 in polyamine oxidase to methionine was previously shown to have no effect on the kinetics of the reductive half-reaction of the enzyme (M. Henderson Pozzi, V. Gawandi, P.F. Fitzpatrick, Biochemistry 48 (2009) 1508-1516). In contrast, the mutation does affect steps in the oxidative half-reaction. The k_cat value is unaffected by the mutation; this kinetic parameter likely reflects product release. At pH 10, the k_cat/K_m value for oxygen is 25-fold lower in the mutant enzyme. The k_cat/K_O2 value is pH-dependent for the wild-type enzyme, decreasing below a pK_a of 7.0, while this kinetic parameter for the mutant enzyme is pH-independent. This is consistent with the neutral form of Lys315 being required for more rapid flavin oxidation. The solvent isotope effect on the k_cat/K_O2 value increases from 1.4 in the wild-type enzyme to 1.9 in the mutant protein, and the solvent inventory changes from linear to bowed. The effects of the mutation can be explained by the lysine orienting the bridging water so that it can accept the proton from the flavin N(5) during flavin oxidation. In the mutant enzyme the lysine amine would be replaced by a water chain.     

Nucleotide recognition by the initiation factor aIF5B: Free energy simulations of a neoclassical GTPase

The GTPase aIF5B is a universally conserved initiation factor that assists ribosome assembly. Crystal structures of its nucleotide complexes, X‐ray(GTP) and X‐ray(GDP), are similar in the nucleotide vicinity, but differ in the orientation of a distant domain IV. This has led to two, contradictory, mechanistic models. One postulates that X‐ray(GTP) and X‐ray(GDP) are, respectively, the active, “ON” and the inactive, “OFF” states; the other postulates that both structures are OFF, whereas the ON state is still uncharacterized. We study GTP/GDP binding using molecular dynamics and a continuum electrostatic free energy method. We predict that X‐ray(GTP) has a ≈ 3 kcal/mol preference to bind GDP, apparently contradicting its assignment as ON. However, the preference arises mainly from a single, nearby residue from the switch 2 motif: Glu81, which becomes protonated upon GTP binding, with a free energy cost of about 4 kcal/mol. We then propose a different model, where Glu81 protonation/deprotonation defines the ON/OFF states. With this model, the X‐ray(GTP):GTP complex, with its protonated Glu81, is ON, whereas X‐ray(GTP):GDP is OFF. The model postulates that distant conformational changes such as domain IV rotation are “uncoupled” from GTP/GDP exchange and do not affect the relative GTP/GDP binding affinities. We analyze the model using a general thermodynamic framework for GTPases. It yields rather precise predictions for the nucleotide specificities of each state, and the state specificities of each nucleotide, which are roughly comparable to the homologues IF2 and aIF2, despite the lack of any conformational switching in the model. © 2012 Wiley Periodicals, Inc.     

Immobilization and properties of carminomycin 4-O-methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.     

Effects of intrastriatal kainic acid injection on [3H]dopamine metabolism in rat striatal slices: evidence for postsynaptic glial cell metabolism by both the type A and B forms of monoamine oxidase

Abstract: Intrastriatal injections of kainic acid (KA) were utilized to investigate the cellular localization of postsynaptic dopamine (DA) metabolism by type A and B monoamine oxidase (MAO) in rat striatum. At 2 days postinjection, maximal degeneration of cholinergic and γ-aminobutyric acid (GABA)ergic neurons was observed and found to be associated with a significant decrease in both type A and B MAO activity. However, over the next 8-day period, when only the process of gliosis appeared to be occurring, a selective return to control of type B MAO activity was seen. When the metabolism of [³H]DA (10^?7 M) was examined in 8-day KA-lesioned rat striatal slices, an increase in [³H]dihydroxyphenylacetic acid (DOPAC) and [³H]homovanillic acid (HVA) formation was observed. The KA-induced elevation of [³H]DOPAC formation (but not [³H]HVA) was abolished by the DA neuronal uptake inhibitor nomifensine. This is consistent with earlier findings suggesting that HVA is formed exclusively within sites external to DA neurons. Experiments with clorgyline and/or deprenyl revealed that the relative roles of type A and B MAO in striatal DA deamination remained unchanged following KA (90% deamination by type A MAO) even though total deamination was substantially enhanced. At high concentrations of [³H]DA (10^?5 M), deamination by type B MAO could be increased to 30% of the total MAO activity; however, this was observed in both control and KA-lesioned striata. These results suggest that KA-sensitive neurons contain type A and/or type B MAO. Moreover, whereas these neurons may metabolize DA, a major portion of postsynaptic DA deamination appears to occur within glial sites of rat striatal tissue. Furthermore, glial cells would appear to contain functionally important quantities of both type A and B MAO.     

Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase

Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher K(m) for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.     

Effect of neurocatin on the activity of monoamine oxidase B in rat brain synaptosomes

Neurocatin, a small (about 2,000 Dalton) neuroregulator isolated from mammalian brain, is a powerful effector of monoamine oxidase B in rat brain synaptosomes. Incubation of intact synaptosomes with neurocatin caused an inhibition of the enzyme dependent on the concentration of neurocatin. This inhibition became statistically significant at a neurocatin concentration of 10 ng/200 l and was significant at all higher neurocatin concentrations. At 40 ng/200 l, neurocatin inhibited monoamine oxidase B activity by about 60%. This inhibitory effect was almost completely abolished by breaking the synaptosomal membrane by hypotonic buffer prior to incubation with neurocatin. In addition, incubation of the synaptosomes in calcium free medium almost completely abolished the inhibitory effect of neurocatin on monoamine oxidase B. The inhibition appeared to involve covalent modification of the enzyme mediated by a neurocatin receptor(s). Measurements of the kinetic parameters of the enzyme showed that 20 ng of neurocatin caused a statistically significant decrease in V_max (by 20%) with no significant change in K_M, compared to controls. Inhibition of monoamine oxidase by neurocatin is potentially of great clinical importance because this enzyme plays a major role in catabolism of the biogenic amines and alterations in its activity is believed to contribute to several neurological disorders.     

类芽孢杆菌来源D-甘露醇氧化酶的性质及制备D-甘露糖的应用

D-甘露糖具有多种功能活性,在食品、医药、农业等行业应用广泛。D-甘露醇氧化酶可以高效地将D-甘露醇转化为D-甘露糖,在D-甘露糖的酶法制备中具有应用潜力。从类芽孢杆菌(Paenibacillus sp.) HGF5中发掘出一个D-甘露醇氧化酶(PsOX),与天蓝链霉菌(Streptomyces coelicolor)来源的D-甘露醇氧化酶(AldO)氨基酸序列相似性为50.94%,分子量约为47.4 kDa,构建了重组表达质粒pET-28a-PsOX并在大肠杆菌BL21(DE3)中表达,PsOX对D-甘露醇的K_m、k_cat/K_m值分别为5.6 mmol/L、0.68 L/(s∙mmol),最适pH和温度分别为7.0和35 ℃,在60 ℃以下保持稳定。PsOX对400 mmol/L D-甘露醇的摩尔转化率为95.2%。利用PsOX与AldO全细胞分别催化73 g/L D-甘露醇,PsOX反应9 h后反应完全,生成70 g/L D-甘露糖,相较于AldO具有更高的催化效率。PsOX作为新型D-甘露糖氧化酶为D-甘露糖的酶法制备提供了依据。     

Nucleophile selectivity in the acyl transfer reaction of a designed enzyme

The acyl transfer reaction of S-glutathionyl benzoate (GSB) is catalyzed by a rationally designed mutant of human glutathione transferase A1-1, A216H. The catalyzed reaction proceeds via the formation of an acyl intermediate and has been studied in the presence of nitrogen, oxygen, and sulfur nucleophiles to determine the selectivity with regards to nucleophile structure. Methanol was previously shown to react with the acyl intermediate and form the corresponding ester, methylbenzoate, under a significant rate enhancement. In the present investigation, the dependence on nucleophile structure and reactivity has been investigated. Ethane thiol gave rise to a larger rate enhancement in the enzyme-catalyzed reaction than ethanol, whereas ethylamine did not increase the reaction rate. The reactivities toward the acyl intermediate of primary and secondary alcohols with similar pKa values depended on the structure of the aliphatic chain, and 1-propanol was the most efficient alcohol. The reactivity of the oxygen nucleophiles was also found to depend strongly on pKa as 2,2,2-trifluoroethanol, with a pKa of 12.4, was the most efficient nucleophile of all that were tested. Saturation kinetics was observed in the case of 1-propanol, indicating a second binding site in the active site of A216H. The nucleophile selectivity of A216H provides the knowledge base needed for the further reengineering of A216H towards alternative substrate specificities.     

Side-chain metabolism of propranolol: involvement of monoamine oxidase and mitochondrial aldehyde dehydrogenase in the metabolism of N-desisopropylpropranolol to naphthoxylactic acid in rat liver

We examined the metabolism of N-desisopropylpropranolol (NDP), which is generated from propranolol (PL) by side-chain N-desisopropylation, to naphthoxylactic acid (NLA) in rat liver. S(-)-NDP (S-NDP) and R(+)-NDP (R-NDP) were enantioselectively metabolized to NLA in isolated rat hepatocytes and in an enzyme reaction system of rat liver mitochondria with cofactor NAD+. Furthermore, the clearance profiles of NDP enantiomers were examined in an enzyme reaction system of rat liver mitochondria without NAD+. The amounts of S-NDP remaining in the incubation medium were similar to those of R-NDP, suggesting that monoamine oxidase (MAO) catalyzes the deamination of NDP to the aldehyde intermediate, but fails to deaminate enantioselectively S-NDP or R-NDP. Cyanamide, a potent inhibitor of aldehyde dehydrogenase (ALDH), markedly decreased the formation of NLA from racemic NDP in the enzyme reaction system of rat liver mitochondria with NAD+. When rat liver cytosol and microsomes were added to this enzyme reaction system, no significant alterations were observed in the amount of NLA generated from racemic NDP. We concluded that MAO deaminates NDP to an aldehyde intermediate, and that mitochondrial ALDH subsequently catalyzes the enantioselective metabolism of the aldehyde intermediate to NLA in rat liver.     

Lipophilicity plays a major role in modulating the inhibition of monoamine oxidase B by 7-substituted coumarins

A series of coumarin derivatives (1-22), bearing at the 7-position ether, ketone, ester, carbamate, or amide functions of varying size and lipophilicity, were synthesized and investigated for their in vitro monoamine oxidase-A and -B (MAO-A and -B) inhibitory activities. Most of the compounds acted preferentially as MAO-B inhibitors, with IC(50) values in the micromolar to low-nanomolar range. A structure-activity-relationship (SAR) study highlighted lipophilicity as an important property modulating the MAO-B inhibition potency of 7-substituted coumarins, as shown by a linear correlation (n=20, r(2)=0.72) between pIC(50) and calculated log P values. The stability of ester-containing coumarin derivatives in rat plasma provided information on factors that either favor (lipophilicity) or decrease (steric hindrance) esterase-catalyzed hydrolysis. Two compounds (14 and 22) were selected to investigate how lipophilicity and enzymatic stability may affect in vivo MAO activities, as assayed ex vivo in rat. The most-potent and -selective MAO-B inhibitor 22 (=7-[(3,4-difluorobenzyl)oxy]-3,4-dimethyl-1-benzopyran-2(2H)-one) within the examined series significantly inhibited (>60%) ex vivo rat-liver and striatal MAO-B activities 1 h after intraperitoneal administration of high doses (100 and 300 mumol kg(-1)), revealing its ability to cross the blood-brain barrier. At the same doses, liver and striatum MAO-A was less inhibited in vivo, somehow reflecting MAO-B selectivity, as assessed in vitro. In contrast, the metabolically less stable derivative 14, bearing an isopropyl ester in the lateral chain, had a weak effect on hepatic MAO-B activity in vivo, and none on striatal MAO-B, but, surprisingly, displayed inhibitory effects on MAO-A in both peripheral and brain tissues.     

Molecular dynamics simulations of the acyl-enzyme and the tetrahedral intermediate in the deacylation step of serine proteases

Despite the availability of many experimental data and some modeling studies, questions remain as to the precise mechanism of the serine proteases. Here we report molecular dynamics simulations on the acyl-enzyme complex and the tetrahedral intermediate during the deacylation step in elastase catalyzed hydrolysis of a simple peptide. The models are based on recent crystallographic data for an acyl-enzyme intermediate at pH 5 and a time-resolved study on the deacylation step. Simulations were carried out on the acyl enzyme complex with His-57 in protonated (as for the pH 5 crystallographic work) and deprotonated forms. In both cases, a water molecule that could provide the nucleophilic hydroxide ion to attack the ester carbonyl was located between the imidazole ring of His-57 and the carbonyl carbon, close to the hydrolytic position assigned in the crystal structure. In the "neutral pH" simulations of the acyl-enzyme complex, the hydrolytic water oxygen was hydrogen bonded to the imidazole ring and the side chain of Arg-61. Alternative stable locations for water in the active site were also observed. Movement of the His-57 side-chain from that observed in the crystal structure allowed more solvent waters to enter the active site, suggesting that an alternative hydrolytic process directly involving two water molecules may be possible. At the acyl-enzyme stage, the ester carbonyl was found to flip easily in and out of the oxyanion hole. In contrast, simulations on the tetrahedral intermediate showed no significant movement of His-57 and the ester carbonyl was constantly located in the oxyanion hole. A comparison between the simulated tetrahedral intermediate and a time-resolved crystallographic structure assigned as predominantly reflecting the tetrahedral intermediate suggests that the experimental structure may not precisely represent an optimal arrangement for catalysis in solution. Movement of loop residues 216-223 and P3 residue, seen both in the tetrahedral simulation and the experimental analysis, could be related to product release. Furthermore, an analysis of the geometric data obtained from the simulations and the pH 5 crystal structure of the acyl-enzyme suggests that since His-57 is protonated, in some aspects, this crystal structure resembles the tetrahedral intermediate.     

Validating computer simulations of enantioselective catalysis; reproducing the large steric and entropic contributions in Candida Antarctica lipase B

The prospect for computer‐aided refinement of stereoselective enzymes is further validated by simulating the ester hydrolysis by the wild‐type and mutants of CalB, focusing on the challenge of dealing with strong steric effects and entropic contributions. This was done using the empirical valence bond (EVB) method in a quantitative screening of the enantioselectivity, considering both k_cat and k_cat/K_M of the R and S stereoisomers. Although the simulations require very extensive sampling for convergence they give encouraging results and major validation, indicating that our approach offers a powerful tool for computer‐aided design of enantioselective enzymes. This is particularly true in cases with large changes in steric effects where alternative approaches may have difficulties in capturing the interplay between steric clashes with the reacting substrate and protein flexibility. Proteins 2014; 82:1387–1399. © 2014 Wiley Periodicals, Inc.     

Identification of the stereochemical requirements in the 4-aryl-2-cycloalkylidenhydrazinylthiazole scaffold for the design of selective human monoamine oxidase B inhibitors

Exploring the effect that substituents on the cycloaliphatic ring had on the inhibitory activity against human monoamine oxidase B of a series of 4-aryl-2-cycloalkylidenhydrazinylthiazoles led to the synthesis of a new series of 2-methylcyclopentyl and 3-methylcyclopentyl derivatives which were tested in vitro as mixtures of diastereoisomers. In fact, due to the presence of a chiral center on the cycloaliphatic ring and a trisubstituted CN bond, they exist as four diastereoisomers ((E)-(R), (E)-(S), (Z)-(R), (Z)-(S)). 4-(2,4-Difluorophenyl)-2-(2-(3-methylcyclopentylidene)hydrazinyl)thiazole was chosen as a model to investigate the influence of stereochemical requirements on the inhibitory activity against hMAO-B of these derivatives after a stereoconservative synthesis and semi-preparative HPLC diastereoseparation. (R)-(Z) isomer of this compound was endowed with a potent and selective hMAO-B inhibition higher than that of reference drugs as also corroborated by molecular modeling studies.     

Restrained molecular dynamics simulations of HIV-1 protease: the first step in validating a new target for drug design

To test the anticorrelated relationship that was recently displayed in conventional molecular dynamics (MD) simulations, several different restrained MD simulations on a wild type and on the V82F/I84V drug-resistant mutant of HIV-1 protease were performed. This anticorrelated relationship refers to the observation that compression of the peripheral ear-to-cheek region of HIV protease (i.e., the elbow of the flap to the fulcrum and the cantilever) occurred as the active site flaps were opening, and, conversely, expansion of that ear-to-cheek region occurred as both flaps were closing. An additional examination of this anticorrelated relationship was necessary to determine whether it can be harnessed in a useful manner. Consequently, six different MD experiments were performed that incorporated pairwise distance restraints in that ear-to-cheek region (i.e., the distance between the alpha-carbons of Gly40 and Gln61 was restrained to either 7.7 or 10.5 A, in both monomers). Pushing the backbones of the ear and the cheek regions away from each other slightly did force the flaps that guard the active site to remain closed in both the wild type and the mutant systems-even though there were no ligands in the active sites. Thus, these restrained MD simulations provided evidence that the anticorrelated relationship can be exploited to affect the dynamic behavior of the flaps that guard the active site of HIV-1 protease. These simulations supported our hypothesis of the mechanism governing flap motion, and they are the first step towards validating that peripheral surface as a new target for drug design.     

Lack of Enantioselectivity in the SULT1A3‐catalyzed Sulfoconjugation of Normetanephrine Enantiomers: An In Vitro and Computational Study

(1R)‐Normetanephrine is the natural stereoisomeric substrate for sulfotransferase 1A3 (SULT1A3)‐catalyzed sulfonation. Nothing appears known on the enantioselectivity of the reaction despite its potential significance in the metabolism of adrenergic amines and in clinical biochemistry. We confronted the kinetic parameters of the sulfoconjugation of synthetic (1R)‐normetanephrine and (1S)‐normetanephrine by recombinant human SULT1A3 to a docking model of each normetanephrine enantiomer with SULT1A3 and the 3′‐phosphoadenosine‐5′‐phosphosulfate cofactor on the basis of molecular modeling and molecular dynamics simulations of the stability of the complexes. The K_M, V_max, and k_cat values for the sulfonation of (1R)‐normetanephrine, (1S)‐normetanephrine, and racemic normetanephrine were similar. In silico models were consistent with these findings as they showed that the binding modes of the two enantiomers were almost identical. In conclusion, SULT1A3 is not substrate‐enantioselective toward normetanephrine, an unexpected finding explainable by a mutual adaptability between the ligands and SULT1A3 through an “induced‐fit model” in the catalytic pocket. Chirality, 25:28‐34, 2012.© 2012 Wiley Periodicals, Inc.     

The nature of tryptophan radicals involved in the long-range electron transfer of lignin peroxidase and lignin peroxidase-like systems: Insights from quantum mechanical/molecular mechanics simulations

A catalytically active tryptophan radical has been demonstrated to be involved in the long-range electron transfer to the heme cofactor of lignin peroxidase (LiP) from Phanerochaete chrysosporium although no direct detection by EPR spectroscopy of the tryptophan radical intermediate has been reported to date. An engineering-based approach has been used to manipulate the microenvironment of the redox-active tryptophan site in LiP and Coprinus cinereus Peroxidase (CiP), allowing the direct evidence of the tryptophan radical species. In light of the newly available EPR experimental data, we performed a quantum mechanical/molecular mechanics computational study to characterize the tryptophan radicals in the above protein matrices as well as in pristine LiP. The nature of the tryptophan radicals is discussed together with the analysis of their environment with the aim of understanding the different behavior of pristine LiP in comparison with that of LiP and CiP variants.     

The rate-limiting step in the folding of the cis-Pro167Thr mutant of TEM-1 β-lactamase is the trans to cis isomerization of a non-proline peptide bond

The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM-1 β-lactamase have been investigated as a function of guanidine hydrochloride concentration. The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166–Thr167 peptide bond, like the Glu166–Pro167, is cis. The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein. In contrast to the two slower phases seen in the refolding of the wild-type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild-type enzyme. The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding. The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans→cis isomerization of the Glu166–Thr167 peptide bond. © 1996 John Wiley & Sons, Inc.     

Kinetics and mechanism of iron exchange in hydroxamate siderophores: Catalysis of the iron(III) transfer from ferrioxamine B to ethylenediaminetetraacetic acid

The oxalate catalyzed iron(III) transfer from a trihydroxamate siderophore ferrioxamine B, [Fe(Hdfb)⁺], to ethylenediaminetetraacetic acid (H₄edta) has been studied spectro-photometrically in weakly acidic aqueous solutions at 298 K and a constant 2.0 M ionic strength maintained by NaClO₄. The results reveal that oxalate is a more efficient catalyst than the so far studied synthetic monohydroxamic acids. Any role of reduction of Fe(Hdfb)⁺ by oxalate in the catalysis has been rejected by the experimentally observed preservation of the oxalate concentration during the reaction time. Therefore, catalysis has been proposed to be a substitution based process. Under our experimental conditions Fe(Hdfb)⁺ is hexacoordinated and addition of oxalate results in the formation of Fe(H₂dfb)(C₂O₄), Fe(H₃dfb)(C₂O₄)⁻₂ and Fe(C₂O₄)³⁻₃. Therefore, catalysis was proposed to be accomplished by the intermediate formation of the ternary and tris(oxalato) complexes. All three complexes react with H₂edta²⁻ to form thermodynamically stable Fe(edta)⁻ as a final reaction product. Whereas the formation of the ternary complexes is fast enough to feature a pre-equilibrium process to the iron exchange reaction, the formation of Fe(C₂O₄)³⁻₃ is slow and is directly involved in the rate determining step of the Fe(edta)⁻ formation. Nonlinear dependencies of the rate constant on the oxalate and the proton concentrations have been observed and a four parallel path mechanism is proposed for the exchange reaction. The rate and equilibrium constants for the various reaction paths were determined from the kinetic and equilibrium study involving the desferrioxamine B- (H₄dfb⁺), oxalate- and proton-concentration variations. The observed proton catalysis was attributed to the fast monoprotonation of ferrioxamine B as well as of the oxalate ligand. The observed catalysis of iron dissociation from the siderophore has been discussed in view of its significance with respect to in vivo microbial iron transport.