Involvement of a flavosemiquinone in the enzymatic oxidation of nitroalkanes catalyzed by 2-nitropropane dioxygenase

2-Nitropropane dioxygenase (EC 1.13.11.32) catalyzes the oxidation of nitroalkanes into their corresponding carbonyl compounds and nitrite. In this study, the ncd-2 gene encoding for the enzyme in Neurospora crassa was cloned, expressed in Escherichia coli, and the resulting enzyme was purified. Size exclusion chromatography, heat denaturation, and mass spectroscopic analyses showed that 2-nitropropane dioxygenase is a homodimer of 80 kDa, containing a mole of non-covalently bound FMN per mole of subunit, and is devoid of iron. With neutral nitroalkanes and anionic nitronates other than propyl-1- and propyl-2-nitronate, for which a non-enzymatic free radical reaction involving superoxide was established using superoxide dismutase, substrate oxidation occurs within the enzyme active site. The enzyme was more specific for nitronates than nitroalkanes, as suggested by the second order rate constant k(cat)/K(m) determined with 2-nitropropane and primary nitroalkanes with alkyl chain lengths between 2 and 6 carbons. The steady state kinetic mechanism with 2-nitropropane, nitroethane, nitrobutane, and nitrohexane, in either the neutral or anionic form, was determined to be sequential, consistent with oxygen reacting with a reduced form of enzyme before release of the carbonyl product. Enzyme-monitored turnover with ethyl nitronate as substrate indicated that the catalytically relevant reduced form of enzyme is an anionic flavin semiquinone, whose formation requires the substrate, but not molecular oxygen, as suggested by anaerobic substrate reduction with nitroethane or ethyl nitronate. Substrate deuterium kinetic isotope effects with 1,2-[(2)H(4)]nitroethane and 1,1,2-[(2)H(3) ethyl nitronate at pH 8 yielded normal and inverse effects on the k(cat)/K(m) value, respectively, and were negligible on the k(cat) value. The k(cat)/K(m) and k(cat) pH profiles with anionic nitronates showed the requirement of an acid, whereas those for neutral nitroalkanes were consistent with the involvement of both an acid and a base in catalysis. The kinetic data reported herein are consistent with an oxidasestyle catalytic mechanism for 2-nitropropane dioxygenase, in which the flavin-mediated oxidation of the anionic nitronates or neutral nitroalkanes and the subsequent oxidation of the enzyme-bound flavin occur in two independent steps.     

2-Nitropropane Dioxygenase from Hansenula mrakii: Re-characterization of the Enzyme and Oxidation of Anionic Nitroalkanes

2-Nitropropane dioxygenase, purified to homogeneity by an improved method from a yeast, Hansenula mrakii, has a molecular weight of 42,000, and consists of a single polypeptide. The enzyme contains 1 mol of FAD per mol of enzyme. The iron protein associated with previous preparations was removed by the present purification procedures. The enzyme catalyzes the oxygenative denitrification of anionic nitroalkanes much more effectively than that of the neutral ones with the optimum pH of 6.5. The Michaelis constants for the anionic substrates are as follows: 2-nitropropane, 1.61 mM; 1-nitropropane, 3.23 mM; nitroethane, 3.13 mM, and 3-nitro-2-butanol, 0.59 mM.     

The nonoxidative conversion of nitroethane to ethylnitronate in Neurospora crassa 2-nitropropane dioxygenase is catalyzed by histidine 196

The deprotonation of nitroethane catalyzed by Neurospora crassa 2-nitropropane dioxygenase was investigated by measuring the formation and release of ethylnitronate formed in turnover as a function of pH and through mutagenesis studies. Progress curves for the enzymatic reaction obtained by following the increase in absorbance at 228 nm over time were visibly nonlinear, requiring a logarithmic approximation of the initial reaction rates for the determination of the kinetic parameters of the enzyme. The pH dependence of the second-order rate constant k cat/ K m with nitroethane as substrate implicates the presence of a group with a p K a of 8.1 +/- 0.1 that must be unprotonated for nitronate formation. Mutagenesis studies suggest that this group is histidine 196 as evident from the inability of a H196N variant form of the enzyme to catalyze the formation of ethylnitronate from nitroethane. Replacement of histidine 196 with asparagine resulted in an approximately 15-fold increase in the k cat/ K m with ethylnitronate as compared to the wild-type, which results from the inability of the mutant enzyme to undergo nonoxidative turnover. The results presented herein are consistent with a branched catalytic mechanism for the enzyme in which the ethylnitronate intermediate formed from the H196-catalyzed deprotonation of nitroethane partitions between release from the active site and oxidative denitrification to yield acetaldehyde and nitrite.     

Electronegativity of aromatic amines as a basis for the development of ground state inhibitors of lysyl oxidase

Benzylamine derivatives containing para substituents of differing electronegativity as well as isomers of aminomethylpyridine have been assessed for their substrate and inhibitor potentials toward lysyl oxidase. Substituted benzylamines with increasingly electronegative para substituents had the lowest KI values and thus were the most effective inhibitors of the oxidation of elastin by lysyl oxidase. The kcat values for these compounds as substrates of lysyl oxidase were also reduced with increasingly electronegative para substituents. Both the Dkcat and D(kcat/Km) kinetic isotope effects decreased with increasingly electronegative p-substituents in [alpha, alpha'-2H]benzylamines. In contrast, there was no Dkcat solvent isotope effect with [2H] H2O while the D(kcat/Km) solvent isotope effect tended to increase with increasingly electronegative p-substituents. These results are consistent with the stabilization of an enzyme-generated substrate carbanion and thus the retardation of substrate oxidation by electronegative substituents. Such ground state stabilization thus can result in compounds with increased potential for the inhibition of the oxidation of protein substrates of lysyl oxidase.     

Kinetic study of carboxypeptidase P-catalyzed reaction. Pressure and temperature dependence of kinetic parameters

Detailed kinetic analyses of carboxypeptidase P-catalyzed reactions were carried out spectrophotometrically using 3-(2-furyl)acryloyl-acylated peptide substrates. The maximum kcat/Km was observed at around pH 3.5 for the synthetic peptide substrates. The kcat/Km value decreased with increasing pH, with an apparent pKa value of 4.43. However, the maximum kcat was observed at neutral pH (pH congruent to 6) and the pKa was 4.49. These apparently different pH profiles for kcat/Km and kcat of this enzyme were due to the decreasing Km value in the acid pH region. The pressure and temperature dependences of these kinetic parameters were also measured. N-Benzoylglycyl-L-phenyllactate (Bz-Gly-OPhLac) gave dependences similar to those of the peptide substrate, suggesting that there is no distinct difference in the catalytic mechanism between the peptide and the ester hydrolyses.     

Purification, Characterization, and Mechanism of a Flavin Mononucleotide-Dependent 2-Nitropropane Dioxygenase from Neurospora crassa

A nitroalkane-oxidizing enzyme was purified to homogeneity from Neurospora crassa. The enzyme is composed of two subunits; the molecular weight of each subunit is approximately 40,000. The enzyme catalyzes the oxidation of nitroalkanes to produce the corresponding carbonyl compounds. It acts on 2-nitropropane better than on nitroethane and 1-nitropropane, and anionic forms of nitroalkanes are much better substrates than are neutral forms. The enzyme does not act on aromatic compounds. When the enzyme reaction was conducted in an ¹⁸O₂ atmosphere with the anionic form of 2-nitropropane as the substrate, acetone (with a molecular mass of 60 Da) was produced. This indicates that the oxygen atom of acetone was derived from molecular oxygen, not from water; hence, the enzyme is an oxygenase. The reaction stoichiometry was 2CH₃CH(NO₂)-CH₃ + O₂→2CH₃COCH₃ + 2HNO₂, which is identical to that of the reaction of 2-nitropropane dioxygenase from Hansenula mrakii. The reaction of the Neurospora enzyme was inhibited by superoxide anion scavengers in the same manner as that of the Hansenula enzyme. Both of these enzymes are flavoenzymes; however, the Neurospora enzyme contains flavin mononucleotide as a prosthetic group, whereas the Hansenula enzyme contains flavin adenine dinucleotide.     

Purification and characterization of the FeII- and alpha-ketoglutarate-dependent xanthine hydroxylase from Aspergillus nidulans

His6-tagged xanthine/alpha-ketoglutarate (alphaKG) dioxygenase (XanA) of Aspergillus nidulans was purified from both the fungal mycelium and recombinant Escherichia coli cells, and the properties of the two forms of the protein were compared. Evidence was obtained for both N- and O-linked glycosylation on the fungus-derived XanA, which aggregates into an apparent dodecamer, while bacterium-derived XanA is free of glycosylation and behaves as a monomer. Immunological methods identify phosphothreonine in both forms of XanA, with phosphoserine also detected in the bacterium-derived protein. Mass spectrometric analysis confirms glycosylation and phosphorylation of the fungus-derived sample, which also undergoes extensive truncation at its amino terminus. Despite the major differences in the properties of these proteins, their kinetic parameters are similar (kcat = 30-70 s-1, Km of alphaKG = 31-50 muM, Km of xanthine approximately 45 muM, and pH optima at 7.0-7.4). The enzyme exhibits no significant isotope effect when [8-2H]xanthine is used; however, it demonstrates a 2-fold solvent deuterium isotope effect. CuII and ZnII potently inhibit the FeII-specific enzyme, whereas CoII, MnII, and NiII are weaker inhibitors. NaCl decreases the kcat and increases the Km of both alphaKG and xanthine. The alphaKG cosubstrate can be substituted with alpha-ketoadipate (9-fold decrease in kcat and 5-fold increase in the Km compared to those of the normal alpha-keto acid), while the alphaKG analogue N-oxalylglycine is a competitive inhibitor (Ki = 0.12 muM). No alternative purines effectively substitute for xanthine as a substrate, and only one purine analogue (6,8-dihydroxypurine) results in significant inhibition. Quenching of the endogenous fluorescence of the two enzyme forms by xanthine, alphaKG, and DHP was used to characterize their binding properties. A XanA homology model was generated on the basis of the structure of the related enzyme TauD (PDB entry 1OS7) and provided insights into the sites of posttranslational modification and substrate binding. These studies represent the first biochemical characterization of purified xanthine/alphaKG dioxygenase.     

Catalytic mechanism of scytalone dehydratase: site-directed mutagenisis, kinetic isotope effects, and alternate substrates.

On the basis of the X-ray crystal structure of scytalone dehydratase complexed with an active center inhibitor [Lundqvist, T., Rice, J., Hodge, C. N., Basarab, G. S., Pierce, J. and Lindqvist, Y. (1994) Structure (London) 2, 937-944], eight active-site residues were mutated to examine their roles in the catalytic mechanism. All but one residue (Lys73, a potential base in an anti elimination mechanism) were found to be important to catalysis or substrate binding. Steady-state kinetic parameters for the mutants support the native roles for the residues (Asn131, Asp31, His85, His110, Ser129, Tyr30, and Tyr50) within a syn elimination mechanism. Relative substrate specificities for the two physiological substrates, scytalone and veremelone, versus a Ser129 mutant help assign the orientation of the substrates within the active site. His85Asn was the most damaging mutation to catalysis consistent with its native roles as a general base and a general acid in a syn elimination. The additive effect of Tyr30Phe and Tyr50Phe mutations in the double mutant is consistent with their roles in protonating the substrate's carbonyl through a water molecule. Studies on a synthetic substrate, which has an anomeric carbon atom which can better stabilize a carbocation than the physiological substrate (vermelone), suggest that His110Asn prefers this substrate over vermelone in order to balance the mutation-imposed weakness in promoting the elimination of hydroxide from substrates. All mutant enzymes bound a potent active-site inhibitor in near 1:1 stoichiometry, thereby supporting their active-site integrity. An X-ray crystal structure of the Tyr50Phe mutant indicated that both active-site waters were retained, likely accounting for its residual catalytic activity. Steady-state kinetic parameters with deuterated scytalone gave kinetic isotope effects of 2.7 on kcat and 4.2 on kcat/Km, suggesting that steps after dehydration partially limit kcat. Pre-steady-state measurements of a single-enzyme turnover with scytalone gave a rate that was 6-fold larger than kcat. kcat/Km with scytalone has a pKa of 7.9 similar to the pKa value for the ionization of the substrate's C6 phenolic hydroxyl, whereas kcat was unaffected by pH, indicating that the anionic form of scytalone does not bind well to enzyme. With an alternate substrate having a pKa above 11, kcat/Km had a pKa of 9.3 likely due to the ionization of Tyr50. The non-enzyme-catalyzed rate of dehydration of scytalone was nearly a billion-fold slower than the enzyme-catalyzed rate at pH 7.0 and 25 degrees C. The non-enzyme-catalyzed rate of dehydration of scytalone had a deuterium kinetic isotope effect of 1.2 at pH 7.0 and 25 degrees C, and scytalone incorporated deuterium from D2O in the C2 position about 70-fold more rapidly than the dehydration rate. Thus, scytalone dehydrates through an E1cb mechanism off the enzyme.     

Leaving group dependence and proton inventory studies of the phosphorylation of a cytoplasmic phosphotyrosyl protein phosphatase from bovine heart.

The kcat and Km values for the bovine heart low molecular weight phosphotyrosyl protein phosphatase catalyzed hydrolysis of 16 aryl phosphate monoesters and of five alkyl phosphate monoesters having the structure Ar(CH2)nOPO3H2 (n = 1-5) were measured at pH 5.0 and 37 degrees C. With the exception of alpha-naphthyl phosphate and 2-chlorophenyl phosphate, which are subject to steric effects, the values of kcat are effectively constant for the aryl phosphate monoesters. This is consistent with the catalysis being nucleophilic in nature, with the existence of a common covalent phosphoenzyme intermediate, and with the breakdown of this intermediate being rate-limiting. In contrast, kcat for the alkyl phosphate monoesters is much smaller and the rate-limiting step for these substrates is interpreted to be the phosphorylation of the enzyme. A single linear correlation is observed for a plot of log (kcat/Km) vs leaving group pKa for both classes of substrates at pH 5.0: log (kcat/Km) = -0.28pKa + 6.88 (n = 19, r = 0.89), indicating a uniform catalytic mechanism for the phosphorylation event. The small change in effective charge (-0.28) on the departing oxygen of the substrate is similar to that observed in the specific acid catalyzed hydrolysis of monophosphate monoanions (-0.27) and is consistent with a strong electrophilic interaction of the enzyme with this oxygen atom in the transition state. The D2O solvent isotope effect and proton inventory experiments indicate that only one proton is "in flight" in the transition state of the phosphorylation process and that this proton transfer is responsible for the reduction of effective charge on the leaving oxygen.     

Mechanistic studies on thrombin catalysis

The kinetic mechanism of the cleavage of four p-nitroanilide (pNA) substrates by human alpha-thrombin has been investigated by using a number of steady-state kinetic techniques. Solvent isotope and viscosity effects were used to determine the stickiness of the substrates at the pH optimum of the reaction; a sticky substrate is defined as one that undergoes catalysis faster than it dissociates from the Michaelis complex. Whereas benzoyl-Arg-pNA could be classified as a nonsticky substrate, D-Phe-pipecolyl-Arg-pNA was very sticky. The other two substrates (tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA) were slightly sticky. The pH profiles of kcat/Km were bell-shaped for all substrates. The pKa values determined from the pH dependence of kcat/Km for benzoyl-Arg-pNA were about 7.5 and 9.1. Similar pKa values were determined from the pH profiles of kcat/Km for tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA and for the binding of the competitive inhibitor N alpha-dansyl-L-arginine-4-methylpiperidine amide. The groups responsible for the observed pKa values were proposed to be His57 and the alpha-amino group of Ile16. The temperature dependence of the pKa values was consistent with this assignment. The pKa values of 6.7 and 8.6 observed in the pH profile of kcat/Km for D-Phe-pipecolyl-Arg-pNA were displaced to lower values than those observed for the other substrates. The displacement of the acidic pKa value could be attributed to the stickiness of this substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of the mechanism of the delta 5-3-ketosteroid isomerase reaction by substrate, solvent, and combined kinetic deuterium isotope effects on wild-type and mutant enzymes

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a conservative tautomeric transfer of the 4 beta-proton to the 6 beta-position using Tyr-14 as a general acid and Asp-38 as a general base [Kuliopulos, A., Mildvan, A. S., Shortle, D., & Talalay, P. (1989) Biochemistry 28, 149]. On deuteration of the 4 beta-position (97.0%) of the substrate, kcat(H)/kcat(4 beta-D) is 6.1 in H2O and 6.3 in D2O. The solvent isotope effect, kcat(H2O)/kcat(D2O), is 1.6 for both the 4 beta-H and 4 beta-D substrates. Mutation of Tyr-55 to Phe lowers kcat 4.3-fold; kcat(H)/kcat/4 beta-D) is 5.3 in H2O and 5.9 in D2O, and kcat(H2O)/kcat(D2O) with the 4 beta-H and 4 beta-D substrates is 1.5 and 1.7, respectively, indicating concerted general acid-base catalysis in either the enolization or the ketonization step of both the wild-type and the Tyr-55----Phe (Y55F) mutant enzymes. An additional slow step occurs with the Y55F mutant. Smaller isotope effects on Km are used to estimate individual rate constants in the kinetic schemes of both enzymes. On deuteration of the 4 alpha-position (88.6%) of the substrate, the secondary isotope effect on kcat/Km corrected for composition is 1.11 +/- 0.02 with the wild-type enzyme and 1.12 +/- 0.02 with the Y55F mutant. These effects decrease to 1.06 +/- 0.01 and 1.07 +/- 0.01, respectively, when the 4 beta-position is also deuterated, thereby establishing these to be kinetic (rather than equilibrium) secondary isotope effects and to involve a proton-tunneling contribution. Deuteration of the 6-position of the substrate (92.0%) produces no kinetic isotope effects on kcat/Km with either the wild-type (1.00 +/- 0.01) or the Y55F mutant (1.01 +/- 0.01) enzyme. Since a change in hybridization from sp3 to sp2 occurs at C-4 only during enolization of the substrate and a change in hybridization at C-6 from sp2 to sp3 occurs only during reketonization of the dienol intermediate, enolization of the substrate constitutes the concerted rate-limiting step. Concerted enolization is consistent with the right angle or antarafacial orientations of Tyr-14 and Asp-38 with respect to the enzyme-bound substrate and with the additive effects on kcat of mutation of these catalytic residues [Kuliopulos, A., Talalay, P., & Mildvan, A. S. (1990) Biophys. J. 57, 39a].     

Comparison of the reaction progress of calcineurin with Mn2+ and Mg2+

Activation of calcineurin by Mn2+ and Mg2+ was compared using a heavy atom isotope analogue of the substrate p-nitrophenyl phosphate (pNPP). Heavy atom isotope effects were measured for Mg2+ activation and compared to published results of the isotope effects with Mn2+ as the activating metal. Isotope effects were measured for the kinetic parameter Vmax/Km at the nonbridging oxygen atoms [18(V/K)nonbridge]; at the position of bond cleavage in the bridging oxygen atom [18(V/K)bridge]; and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The isotope effects increased in magnitude upon changing from an optimal pH to a nonoptimal pH; the 18(V/K)bridge effect increased from 1.0154 (+/-0.0007) to 1.0198 (+/-0.0002), and the 15(V/K) effect increased from 1.0018 (+/-0. 0002) to 1.0021 (+/-0.0003). The value for 18(V/K)nonbridge is 0. 9910 (+/-0.0003) at pH 7.0. As with Mn2+, the 18(V/K)nonbridge isotope effect indicated that the dianion was the substrate for catalysis, and that a dissociative transition state was operative for the phosphoryl transfer. Comparison to results for Mn2+ activation suggested that chemistry was more rate-limiting with Mg2+ than with Mn2+. Changing the activating metal concentration showed opposite trends with increasing Mg2+ increasing the commitment factor and seemingly making the chemistry less rate-limiting. The influence of viscosity was evaluated as well to gauge the role of chemistry. The activation of calcineurin-catalyzed hydrolysis of pNPP1 by Mg2+ or Mn2+ at pH 7.0 was compared in the presence of viscogens, glycerol and poly(ethylene glycol). Increasing glycerol caused different effects with the two activators. With Mn2+ as the activator, calcineurin activity showed a normal response with kcat and kcat/Km decreasing with viscosity. There was an inverse response with Mg2+ as the activator as values of kcat/Km increased with viscosity. From values of the normalized kcat/Km with Mn2+, the chemistry was found to be partially rate-limiting, consistent with previous heavy atom isotope studies (22). The effect observed for Mg2+ seems consistent with a change in the rate-limiting step for the two different metals at pH 7.0.     

Studies on the aminopeptidase activity of rat cathepsin H.

Three synthetic substrates H-Arg-NH-Mec, Bz-Arg-NH-Mec and H-Cit-NH-Mec (Bz, Benzoyl; NH-Mec, 4-methylcoumaryl-7-amide; Cit, citrulline) were used to characterize specificity requirements for the P1-S1 interaction of cathepsin H from rat liver. From rapid equilibrium kinetic studies it was shown that Km, kcat and the specificity constants kcat/Km are quite similar for substrates with a free alpha-amino group. In contrast, a 25-fold decrease of kcat/Km was observed for the N-terminal-blocked substrate Bz-Arg-NH-Mec. The activation energies for H-Arg-NH-Mec and Bz-Arg-NH-Mec were determined to be 37 kJ/mol and 55 kJ/mol, respectively, and the incremental binding energy delta delta Gb of the charged alpha-amino group was estimated to -8.1 kJ/mol at pH 6.8. The shown preference of cathepsin H for the unblocked substrates H-Arg-NH-Mec and H-Cit-NH-Mec was further investigated by inspection of the pH dependence of kcat/Km. The curves of the two substrates with a charged alpha-amino group showed identical bell-shaped profiles which both exhibit pKa1 and pKa2 values of 5.5 and 7.4, respectively, at 30 degrees C. The residue with a pKa1 of 5.5 in the acid limb of the activity profile of H-Arg-NH-Mec was identified by its ionization enthalpy delta Hion = 21 kJ/mol as a beta-carboxylate or gamma-carboxylate of the enzyme, whereas the residue with a pKa2 of 7.4 was assigned to the free alpha-amino group of the substrate with a delta Hion of 59 kJ/mol. Bz-Arg-NH-Mec showed a different pH-activity profile with a pKa1 of 5.4 and a pKa2 of 6.6 at 30 degrees C. Cathepsin H exhibits no preference for a basic P1 side chain as has been shown by the similar kinetics of H-Arg-NH-Mec and the uncharged, isosteric substrate H-Cit-NH-Mec. In summary, specific interactions of an anionic cathepsin H active site residue with the charged alpha-amino group of substrates caused transition state stabilization which proves the enzyme to act preferentially as an aminopeptidase.     

Functional role of residues in the helix B' region of cytochrome P450 2B1

While several flavoproteins will oxidize nitroalkanes in addition to their physiological substrates, nitroalkane oxidase (NAO) is the only one which does not require the anionic nitroalkane. This, in addition to the induction of NAO by nitroethane seen in Fusarium oxysporum, suggests that oxidation of a nitroaliphatic species is the physiological role of the enzyme. Mechanistic studies of the reaction with nitroethane as substrate have established many of the details of the enzymatic reaction. The enzyme is unique in being the only flavoprotein to date for which a carbanion is definitively established as an intermediate in catalysis. Recent structural analyses show that NAO is homologous to the acyl-CoA dehydrogenase and acyl-CoA oxidase families of enzymes. In NAO, the glutamate which acts as the active site base in the latter enzymes is replaced by an aspartate.     

Nitronate monooxygenase, a model for anionic flavin semiquinone intermediates in oxidative catalysis

Nitronate monooxygenase (NMO), formerly referred to as 2-nitropropane dioxygenase, is an FMN-dependent enzyme that uses molecular oxygen to oxidize (anionic) alkyl nitronates and, in the case of the enzyme from Neurospora crassa, (neutral) nitroalkanes to the corresponding carbonyl compounds and nitrite. Over the past 5 years, a resurgence of interest on the enzymology of NMO has driven several studies aimed at the elucidation of the mechanistic and structural properties of the enzyme. This review article summarizes the knowledge gained from these studies on NMO, which has been emerging as a model system for the investigation of anionic flavosemiquinone intermediates in the oxidative catalysis of organic molecules, and for the effect that branching of reaction intermediates has on both the kinetic parameters and isotope effects associated with enzymatic reactions. A comparison of the catalytic mechanism of NMO with other flavin-dependent enzymes that oxidize nitroalkane and nitronates is also presented.     

On the contribution of the positively charged headgroup of choline to substrate binding and catalysis in the reaction catalyzed by choline oxidase

Recent kinetic studies established that the positive charge on the trimethylammonium group of choline plays an important role in substrate binding and specificity in the reaction catalyzed by choline oxidase. In the present study, pH and solvent viscosity effects with the isosteric analogue of choline 3,3-dimethyl-butan-1-ol have been used to further dissect the contribution of the substrate positive charge to substrate binding and catalysis in the reaction catalyzed by choline oxidase. Both the kcat and kcat/Km values with 3,3-dimethyl-butan-1-ol increased to limiting values that were approximately 3- and approximately 400-times lower than those observed with choline, defining pKa values that were similar to the thermodynamic pKa value of approximately 7.5 previously determined. No effects of increased solvent viscosity were observed on the kcat and kcat/Km values with the substrate analogue at pH 8, suggesting that the chemical step of substrate oxidation is fully rate-limiting for the overall turnover and the reductive half-reaction in which the alcohol substrate is oxidized to the aldehyde. The kcat/Km value for oxygen determined with the substrate analogue was pH-independent in the pH range from 6 to 10, with an average value that was approximately 75-times lower than that previously determined with choline as substrate. These data are consistent with the positive charge headgroup of choline playing important roles for substrate binding and flavin oxidation, with minimal contribution to substrate oxidation.     

Steady-state kinetic characterization of evolved biphenyl dioxygenase, which acquired novel degradation ability for benzene and toluene

Biphenyl dioxygenase (Bph Dox) catalyzes initial oxygenation in the bacterial biphenyl degradation pathway. Bph Dox in Pseudomonas pseudoalcaligenes KF707 is a Rieske type three-component enzyme in which a large subunit (encoded by the bphA1 gene) plays an important role in the substrate specificity of Bph Dox. Steady-state kinetic assays using purified enzyme components demonstrated that KF707 Bph Dox had a kcat/Km of 33.1 x 10(3) (M(-1) s(-1)) for biphenyl. Evolved 1072 Bph Dox generated by the process of DNA shuffling (Suenaga, H. et al., J. Bacteriol., 184, 3682-3688 (2002)) exhibited enhanced degradation activity not only for biphenyl (kcat/Km of 62.2 x 10(3) [M(-1) s(-1)]) but also for benzene and toluene, compounds that are rarely attacked by KF707 Bph Dox. These results suggest that evolved 1072 Bph Dox acquires higher affinities and catalytic efficiencies for various substrates than the original KF707 enzyme.     

The pH-dependence and group modification of beta-lactamase I.

The pH-dependence of the kinetic parameters for the hydrolysis of the beta-lactam ring by beta-lactamase I (penicillinase, EC 3.5.2.6) was studied. Benzylpenicillin and ampicillin (6-[D(-)-alpha-aminophenylacetamido]penicillanic acid) were used. Both kcat. and kcat./Km for both substrates gave bell-shaped plots of parameter versus pH. The pH-dependence of kcat./Km for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin). For benzylpenicillin both kcat. and kcat./Km depended on pH in exactly the same way. The value of Km for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme's catalytically important groups does not affect binding of this substrate. The pH-dependence of kcat. for ampicillin differed, however, presumably because of the polar group in the side chain. The hypothesis that the pK5 group is a carboxyl group was tested. Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward's reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate. These findings tend to support the idea that a carboxylate group plays a part in the action of beta-lactamase I.     

Construction of an altered proton donation mechanism in Escherichia coli dihydrofolate reductase

We have explored the substrate protonation mechanism of Escherichia coli dihydrofolate reductase by changing the location of the proton donor. A double mutant was constructed in which the proton donor of the wild-type enzyme, aspartic acid-27, has been changed to serine and simultaneously an alternative proton donor, glutamic acid, has replaced threonine at position 113. The active site of the resulting variant enzyme molecule should therefore somewhat resemble that proposed for the R67 plasmid-encoded dihydrofolate reductase [Matthews, D. A., Smith, S. L., Baccanari, D. P., Burchall, J. J., Oatley, S. J., & Kraut, J. (1986) Biochemistry 25, 4194]. At pH 7, the double-mutant enzyme has a 3-fold greater kcat and an unchanged Km(dihydrofolate) as compared with the single-mutant Asp-27----Ser enzyme described previously [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science (Washington, D.C.) 231, 1123]. Additionally, its activity vs pH profiles together with observed deuterium isotope effects, suggest that catalysis depends on an acidic group with a pKa of 8. It is concluded that the dihydropteridine ring of a bound substrate molecule can indeed be protonated by a glutamic acid side chain at position 113 (instead of an aspartic acid side chain at position 27), but with greatly decreased efficiency: at pH 7, the double mutant still has a 25-fold lower kcat (1.2 s-1) and a 2900-fold lower kcat/km(dihydrofolate) (8.6 X 10(3) s-1 M-1) than the wild-type enzyme.     

PPIase catalysis by human FK506-binding protein proceeds through a conformational twist mechanism.

FK506-binding protein (FKBP) catalyzes the cis-trans isomerization of the peptidyl-prolyl amide bond (the PPIase reaction) and is the major intracellular receptor for the immunosuppressive drugs FK506 and rapamycin. One mechanism proposed for catalysis of the PPIase reaction requires attack of an enzyme nucleophile on the carbonyl carbon of the isomerized peptide bond. An alternative mechanism requires conformational distortion of the peptide bond with or without assistance by an enzyme hydrogen bond donor. We have determined the kinetic parameters of the human FKBP-catalyzed PPIase reaction. At 5 degrees C, the isomerization of Suc-Ala-Leu-Pro-Phe-pNA proceeds in 2.5% trifluorethanol with kcat = 600 s-1, Km = 0.5 mM and kcat/Km = 1.2 x 10(6) M-1s-1. The kcat/Km shows little pH dependence between 5 and 10. A normal secondary deuterium isotope effect is observed on both kcat and kcat/Km. To investigate dependence on enzyme nucleophiles and proton donors, we have replaced eight potential catalytic residues with alanine by site-directed mutagenesis. Each FKBP variant efficiently catalyzes the PPIase reaction. Taken together, these data support an unassisted conformational twist mechanism with rate enhancement due in part to desolvation of the peptide bond at the active site. Fluorescence quenching of the buried tryptophan 59 residue by peptide substrate suggests that isomerization occurs in a hydrophobic environment.