Reaction mechanism of fluoroacetate dehalogenase from Moraxella sp. B

Fluoroacetate dehalogenase (EC 3.8.1.3) catalyzes the dehalogenation of fluoroacetate and other haloacetates. The amino acid sequence of fluoroacetate dehalogenase from Moraxella sp. B is similar to that of haloalkane dehalogenase (EC 3.8.1.5) from Xanthobacter autotrophicus GJ10 in the regions around Asp-105 and His-272, which correspond to the active site nucleophile Asp-124 and the base catalyst His-289 of the haloalkane dehalogenase, respectively (Krooshof, G. H., Kwant, E. M., Damborsky, J., Koca, J., and Janssen, D. B. (1997) Biochemistry 36, 9571-9580). After multiple turnovers of the fluoroacetate dehalogenase reaction in H218O, the enzyme was digested with trypsin, and the molecular masses of the peptide fragments formed were measured by ion-spray mass spectrometry. Two 18O atoms were shown to be incorporated into the octapeptide, Phe-99-Arg-106. Tandem mass spectrometric analysis of this peptide revealed that Asp-105 was labeled with two 18O atoms. These results indicate that Asp-105 acts as a nucleophile to attack the alpha-carbon of the substrate, leading to the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule on the carbonyl carbon atom. A His-272 --> Asn mutant (H272N) showed no activity with either fluoroacetate or chloroacetate. However, ion-spray mass spectrometry revealed that the H272N mutant enzyme was covalently alkylated with the substrate. The reaction of the H272N mutant enzyme with [14C]chloroacetate also showed the incorporation of radioactivity into the enzyme. These results suggest that His-272 probably acts as a base catalyst for the hydrolysis of the covalent ester intermediate.     

Unusual mechanism of oxygen atom transfer and product rearrangement in the catalytic reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase

The oxygenation reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with the substrate, MHPC, was investigated. Two oxygenated flavin intermediates C(4a)-hydroperoxy flavin and C(4a)-hydroxy flavin were found, implying that the enzyme functions similarly to flavoprotein hydroxylases. This finding is supported by the results of independent oxygen-18 tracer experiments, which showed that one atom of oxygen from 18O2 and one atom of oxygen from H218O are incorporated in the product. MHPC oxygenase normally catalyzes both the oxygenation and the hydrolytic ring opening of the pyridine ring of MHPC to yield the acyclic compound, alpha-(N-acetylaminomethylene)succinic acid. Using 5-hydroxynicotinic acid (5HN), which has no 2-methyl group, we tested whether the hydrolytic reaction was due to the presence of the 2-methyl group on MHPC (that prevented rearomatization of the initial product) or to the specific properties of MHPC oxygenase. Product analysis of the enzymatic reaction of 5HN and MHPC oxygenase shows that the enzyme catalyzes the hydroxylation and subsequent hydrolysis of the hydroxylated substrate to yield an acyclic product. The investigation of the oxygenation reaction demonstrates that the enzyme uses the same mechanism to catalyze the 5HN reaction as it does in the MHPC reaction.     

Paracatalytic inactivation of L-2-haloacid dehalogenase from Pseudomonas sp. YL by hydroxylamine. Evidence for the formation of an ester intermediate

Asp10 of L-2-haloacid dehalogenase from Pseudomonas sp. YL was proposed to act as a nucleophile to attack the alpha-carbon of L-2-haloalkanoic acids to form an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon (Liu, J.-Q, Kurihara, T., Miyagi, M., Esaki, N., and Soda, K. (1995) J. Biol. Chem. 270, 18309-18312). We have found that the enzyme is paracatalytically inactivated by hydroxylamine in the presence of the substrates monochloroacetate and L-2-chloropropionate. Ion spray mass spectrometry demonstrated that the molecular mass of the enzyme inactivated by hydroxylamine during the dechlorination of monochloroacetate is about 74 Da greater than that of the native enzyme. To determine the increase of the molecular mass more precisely, we digested the inactivated enzyme with lysyl endopeptidase and measured the molecular masses of the peptide fragments. The molecular mass of the hexapeptide Gly6-Lys11 was shown to increase by 73 Da. Tandem mass spectrometric analysis of this peptide revealed that the increase is due to a modification of Asp10. When the enzyme was paracatalytically inactivated by hydroxylamine during the dechlorination of L-2-chloropropionate, the molecular mass of the hexapeptide was 87 Da higher. Hydroxylamine is proposed to attack the carbonyl carbon of the ester intermediate and form a stable aspartate beta-hydroxamate carboxyalkyl ester residue in the inactivated enzyme.     

Crystal structures of reaction intermediates of L-2-haloacid dehalogenase and implications for the reaction mechanism

Crystal structures of L-2-haloacid dehalogenase from Pseudomonas sp. YL complexed with monochloroacetate, L-2-chlorobutyrate, L-2-chloro-3-methylbutyrate, or L-2-chloro-4-methylvalerate were determined at 1.83-, 2.0-, 2.2-, and 2.2-A resolutions, respectively, using the complex crystals prepared with the S175A mutant, which are isomorphous with those of the wild-type enzyme. These structures exhibit unique structural features that correspond to those of the reaction intermediates. In each case, the nucleophile Asp-10 is esterified with the dechlorinated moiety of the substrate. The substrate moieties in all but the monochloroacetate intermediate have a D-configuration at the C2 atom. The overall polypeptide fold of each of the intermediates is similar to that of the wild-type enzyme. However, it is clear that the Asp-10-Ser-20 region moves to the active site in all of the intermediates, and the Tyr-91-Asp-102 and Leu-117-Arg-135 regions make conformational changes in all but the monochloroacetate intermediates. Ser-118 is located near the carboxyl group of the substrate moiety; this residue probably serves as a binding residue for the substrate carboxyl group. The hydrophobic pocket, which is primarily composed of the Tyr-12, Gln-42, Leu-45, Phe-60, Lys-151, Asn-177, and Trp-179 side chains, exists around the alkyl group of the substrate moiety. This pocket may play an important role in stabilizing the alkyl group of the substrate moiety through hydrophobic interactions, and may also play a role in determining the stereospecificity of the enzyme. Moreover, a water molecule, which is absent in the substrate-free enzyme, is present in the vicinities of the carboxyl carbon of Asp-10 and the side chains of Asp-180, Asn-177, and Ala-175 in each intermediate. This water molecule may hydrolyze the ester intermediate and its substrate. These findings crystallographically demonstrate that the enzyme reaction proceeds through the formation of an ester intermediate with the enzyme's nucleophile Asp-10.     

The key step in chlorophyll breakdown in higher plants. Cleavage of pheophorbide a macrocycle by a monooxygenase

Chlorophyll breakdown in green plants is a long-standing biological enigma. Recent work has shown that pheophorbide a (Pheide a) derived from chlorophyll (Chl) is converted oxygenolytically into a primary fluorescent catabolite (pFCC-1) via a red Chl catabolite (RCC) intermediate. RCC, the product of the ring cleavage reaction catalyzed by Pheide a oxygenase, which is suggested to be the key enzyme in Chl breakdown in green plants, is converted into pFCC-1 by a reductase. In the present study, an in vitro assay comprising 18O2 Pheide a oxygenase and RCC reductase yielded labeled pFCC-1. Fast atom bombardment-mass spectrometric analysis of the purified pFCC-1 product revealed that only one of the two oxygen atoms newly introduced into Pheide a in the course of the cleavage reaction is derived from molecular oxygen. Analysis of the fragment ions located the oxygen atom derived from molecular oxygen on the formyl group of pyrrole B. This finding demonstrates that the cleavage of Pheide a in vascular plants is catalyzed by a monooxygenase. Chlorophyll breakdown is therefore indicated to be mechanistically related in higher plants and in the green alga Chlorella protothecoides.     

Accurate peptide sequencing by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Partial 18O-labeling of peptides has been applied to post-source decay experiments in a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The ions which originate from the carboxyl terminus of a peptide partially retain 18O atoms which have readily been incorporated into the C-terminal carboxyl groups during enzymatic hydrolysis in a buffer containing 40 atom percent H218O. The isotopic resolution of singly charged precursor ions and their product ions obtained for peptides up to ca. 2800 Da has been achieved using the delayed extraction method, which permits the rapid identification and assignment of the 18O-labeled and non-labeled ion species in the PSD spectra. The results obtained from several 18O-labeled peptides, derived from an enzymatic digest, demonstrate the accuracy and reproducibility of the present method, which will be in widespread use for protein identification via database searching or for investigations of totally unknown proteins.     

Energetics of triosephosphate isomerase: the nature of the proton transfer between the catalytic base and solvent water

The isomerization of specifically deuterium-labeled [1(R)-2H5dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate, catalyzed by the enzyme triosephosphate isomerase, has been studied. It is shown that the extent of transfer of the 2H label from the substrate to the product D-glyceraldehyde 3-phosphate is (after complete reaction) the same as that of the corresponding transfer of 3H. The absence of an isotope effect shows that the exchange process of the tstopically labeled enzyme carboxyl group, -COOL H2O leads to -COOH + LOH, does not tnvolve a rate-limiting transition state in which L is the flight. Possible modes for the nature of the ionization of -COOL in 1H2O are discussed.     

Endogenous 7-oxocholesterol is an enzymatic product: characterization of 7 alpha-hydroxycholesterol dehydrogenase activity of hamster liver microsomes

Previously, we described a new metabolite derived from endogenous cholesterol in the presence of hamster liver microsomal protein and NADPH (Song et al., 1991, Biochem. Pharmacol. 41, 1439-1447). Through gas chromatography/mass spectral analysis of the metabolite and its methoxime-3-dimethyl-t-butylsilyl ether derivative, this metabolite has been definitively identified as 7-oxocholesterol. Isotope incorporation experiments using molecular 18O2 demonstrated that no oxygen atoms from molecular oxygen were incorporated into the product, 7-oxocholesterol, when 7 alpha-hydroxycholesterol was used as substrate. In contrast, one atom of 18O was incorporated into cholesterol from 18O2 during its metabolism to form 7 alpha-hydroxycholesterol. Formation of 7-oxocholesterol was dependent upon the presence of NADP+, 7 alpha-hydroxycholesterol, and hamster liver microsomes. This enzyme appears to be a membrane-bound protein and its activity was most abundant in liver microsomal fractions and to a lesser extent in mitochondrial fractions; little or no activity was observed in nuclei or cytosol. The enzyme activity was present in highest content in the livers of hamsters and was also observed in human and bovine liver microsomes, but not those of mouse, rabbit, or rat. The reaction was inhibited by 2'-AMP, but not by anti-NADPH:cytochrome-P450 oxidoreductase globulin, carbon monoxide, metyrapone, nor miconazole. In contrast to the previously characterized 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity, NAD+ did not serve as an effective cofactor for 7-oxocholesterol formation. The ability of NADPH to partially serve as a cofactor in this reaction was shown to be due to a high NADPH-oxidase activity of hamster liver microsomes, thereby providing sufficient NADP+ to serve as the oxidizing pyridine nucleotide for the reaction. These results document the existence of a non-P450, NADP(+)-dependent 7 alpha-hydroxycholesterol dehydrogenase in liver microsomes which catalyzes this reaction. The product, 7-oxocholesterol, is produced enzymatically in the livers of hamsters and other mammals and may regulate bile acid metabolism or other processes due to its action as an oxysterol.     

Crystal structures of nucleic acid complexes of ribonuclease U2

Crystal structures of adenine-specific Ustilago sphaerogena ribonuclease U2 complexed with the substrate analogues, d(ApG), d(ApGpG), and d(ApGpC), with the intermediate analogue, 2',3'-O-isopropylidene-adenosine, and with the product, 3'-AMP, have been determined. In each structure, the adenine base is recognized by the enzyme with four hydrogen-bonds. In the substrate analogue structures, the second base of guanine is sandwiched between His 101 and Tyr 107 side-chains, and forms two hydrogen-bonds with Tyr 107 O and Asp 108 O delta 1 atoms. The third base of the trinucleotides is in van der Waals interaction with the Tyr 78 side-chain. The phosphate group between the second and third nucleosides forms two hydrogen-bonds with the side chains of Asp 37 and Tyr 78. Oxygen atoms of the scissile phosphate group are involved in interactions with catalytic residues of Tyr 39, His 41, Glu 62, Arg 85, and His 101. These interactions indicate that either His 41 or Glu 62 acts as a general base and His 101 acts as a general acid in the first step of RNA hydrolysis.     

Site-directed mutagenesis and oxygen isotope incorporation studies of the nucleophilic aspartate of haloalkane dehalogenase

Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in a broad range of halogenated aliphatic compounds. The X-ray structure suggests that Asp124, which is located close to an internal cavity, carries out a nucleophilic attack on the C alpha of the substrate, releasing the halogen. To study the mechanism of hydrolysis, this aspartate residue was mutated to alanine, glycine, or glutamate. The mutant enzymes showed no activity toward 1,2-dichloroethane and 1,2-dibromoethane. Incubation of purified wild-type dehalogenase with 1,2-dichloroethane in the presence of H2(18)O resulted in the incorporation of 18O in 2-chloroethanol and in the carboxylate group of Asp124. This shows that the reaction proceeds by covalent catalysis with the formation of an alkyl-enzyme intermediate that is hydrolyzed by attack of solvent water on the carbonyl carbon of Asp124. On the basis of amino acid sequence similarity between haloalkane dehalogenase and epoxide hydrolases, it is proposed that a conserved aspartate residue is also involved in covalent catalysis by the latter enzymes.     

The axial tyrosinate Fe3+ ligand in protocatechuate 3,4-dioxygenase influences substrate binding and product release: evidence for new reaction cycle intermediates

The essential active site Fe3+ of protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown that Tyr447 is dissociated from the Fe3+ in the anaerobic 3,4-PCD complex with protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here by site-directed mutation of this residue to His (Y447H), the first such mutation reported for an aromatic ring cleavage dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181) is essentially unchanged from that of the native enzyme outside of the active site region. The side chain position of His447 is stabilized by a His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827 appears to be replaced by a CO32-, thereby retaining the overall charge neutrality and coordination number of the Fe3+ center. Quantitative metal and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than that of the native enzyme. Single-turnover kinetic analysis of the Y447H-catalyzed reaction reveals that slow substrate binding accounts for the decreased kcat. Three new kinetically competent intermediates in this process are revealed. Similarly, the product dissociation from Y447H is slow and occurs in two resolved steps, including a previously unreported intermediate. The final E.PCA complex (ES4) and the putative E.product complex (ESO2*) are found to have optical spectra that are indistinguishable from those of the analogous intermediates of the wild-type enzyme cycle, while all of the other observed intermediates have novel spectra. Once the E.S complex is formed, reaction with O2 is fast. These results suggest that dissociation of Tyr447 occurs during turnover of 3,4-PCD and is important in the substrate binding and product release processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex by either dissociation or mutagenesis, the O2 attack and insertion steps proceed efficiently, suggesting that Tyr447 does not have a large role in this phase of the reaction. This study demonstrates a novel role for Tyr in a biological system and allows evaluation and refinement of the proposed Fe3+ dioxygenase mechanism.     

Kinetic determination of the fast exchanging substrate water molecule in the S3 state of photosystem II

In a previous communication we showed from rapid isotopic exchange measurements that the exchangeability of the substrate water at the water oxidation catalytic site in the S3 state undergoes biphasic kinetics although the fast phase could not be fully resolved at that time [Messinger, J., Badger, M., and Wydrzynski, T. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3209-3213]. We have since improved the time resolution for these measurements by a further factor of 3 and report here the first detailed kinetics for the fast phase of exchange. First-order exchange kinetics were determined from mass spectrometric measurements of photogenerated O2 as a function of time after injection of H218O into spinach thylakoid samples preset in the S3 state at 10 degreesC. For measurements made at m/e = 34 (i. e., for the mixed labeled 16,18O2 product), the two kinetic components are observed: a slow component with k1 = 2.2 +/- 0.1 s-1 (t1/2 approximately 315 ms) and a fast component with k2 = 38 +/- 4 s-1 (t1/2 approximately 18 ms). When the isotopic exchange is measured at m/e = 36 (i.e., for the double labeled 18,18O2 product), only the slow component (k1) is observed, clearly indicating that the substrate water undergoing slow isotopic exchange provides the rate-limiting step in the formation of the double labeled 18,18O2 product. When the isotopic exchange is measured as a function of temperature, the two kinetic components reveal different temperature dependencies in which k1 increases by a factor of 10 over the range 0-20 degreesC while k2 increases by only a factor of 3. Assuming simple Arrhenius behavior, the activation energies are estimated to be 78 +/- 10 kJ mol-1 for the slow component and 39 +/- 5 kJ mol-1 for the fast component. The different kinetic components in the 18O isotopic exchange provide firm evidence that the two substrate water molecules undergo separate exchange processes at two different chemical sites in the S3 state, prior to the O2 release step (t1/2 approximately 1 ms at 20 degreesC). The results are discussed in terms of how the substrate water may be bound at two separate metal sites.     

Crossreactivity, efficiency and catalytic specificity of an esterase-like antibody

The antibody D2.3 catalyzes the hydrolysis of several p-nitrobenzyl and p-nitrophenyl esters with significant rate enhancement; product inhibition is observed with the former compounds but not with the latter. Whereas enzyme specificity has been extensively studied by X-ray crystallography, structural data on catalytic antibodies have thus far related only to one of the reactions they catalyze. To investigate the substrate specificity and the substrate relative to product selectivity of D2.3, we have determined the structures of its complexes with two p-nitrophenyl phosphonate transition state analogs (TSAs) and with the reaction product, p-nitrophenol. The complexes with these TSAs, determined at 1.9 A resolution, and that with p-nitrobenzyl phosphonate determined previously, differ mainly by the locations and conformations of the ligands. Taken together with kinetic data, the structures suggest that a hydrogen bond to an atom of the substrate distant by eight covalent bonds from the carbonyl group of the hydrolyzed ester bond contributes to catalytic efficiency and substrate specificity. The structure of Fab D2.3 complexed with p-nitrophenol was determined at 2.1 A resolution. Release of p-nitrophenol is facilitated due to the unfavourable interaction of the partial charge of the nitro group of p-nitrophenolate with the hydrophobic cavity where it is located, and to the absence of a direct hydrogen bond between the product and the Fab. Catalytic specificity and the manner of product release are both affected by interactions with substrate atoms remote from the reaction center that were not programmed in the design of the TSA used to elicit this antibody. Selection of a catalytic antibody that makes use of TSA unprogrammed features has been made practical because of the screening for catalytic efficiency incorporated in the procedure used to obtain it.     

The substrate specificity of cytochrome P450cam

The catalytic turnover of xenobiotics by cytochrome P450cam results in both the formation of organic metabolites and the uncoupled production of H2O2, and H2O. Previous studies have shown that a receptor-constrained three-dimensional screening program (DOCK) can be used to identify potential ligands (ergo substrates) for the enzyme (De Voss, J. J.; Sibbesen, O.; Zhang, Z.; Ortiz de Montellano, P. R. J. Am. Chem. Soc. 1997, 119, 5489). A new set of 10 compounds has now been examined to further test the substrate specificity of P450cam and the ability of DOCK to identify substrates for this enzyme. The results expand the known specificity of P450cam and define limitations in the use of DOCK to predict its substrate specificity.     

Isotope exchange studies on the Escherichia coli selenophosphate synthetase mechanism

Selenophosphate synthetase, the Escherichia coli selD gene product, is a 37-kDa protein that catalyzes the synthesis of selenophosphate from ATP and selenide. In the absence of selenide, ATP is converted quantitatively to AMP and two orthophosphates in a very slow partial reaction. A monophosphorylated enzyme derivative containing the gamma-phosphoryl group of ATP has been implicated as an intermediate from the results of positional isotope exchange studies. Conservation of the phosphate bond energy in the final selenophosphate product is indicated by its ability to phosphorylate alcohols and amines to form O-phosphoryl- and N-phosphoryl-derivatives. To further probe the mechanism of action of selenophosphate synthetase, isotope exchange studies with [8-14C]ADP or [8-14C]AMP and unlabeled ATP were carried out, and 31P NMR analysis of reaction mixtures enriched in H218O was performed. A slow enzyme-catalyzed exchange of ADP with ATP observed in the absence of selenide implies the existence of a phosphorylated enzyme and further supports an intermediary role of ADP in the reaction. Under these conditions ADP is slowly converted to AMP. Incorporation of 18O from H218O exclusively into orthophosphate in the overall selenide-dependent reaction indicates that the beta-phosphoryl group of the enzyme-bound ADP is attacked by water with liberation of orthophosphate and formation of AMP. Based on these results and the failure of the enzyme to catalyze an exchange of labeled AMP with ATP, the existence of a pyrophosphorylated enzyme intermediate that was postulated earlier can be excluded.     

Enzymes harboring unnatural amino acids: mechanistic and structural analysis of the enhanced catalytic activity of a glutathione transferase containing 5-fluorotryptophan

The catalytic characteristics and structure of the M1-1 isoenzyme of rat glutathione (GSH) transferase in which all four tryptophan residues in each monomer are replaced with 5-fluorotryptophan are described. The fluorine-for-hydrogen substitution does not change the interaction of the enzyme with GSH even though two tryptophan residues (Trp7 and Trp45) are involved in direct hydrogen-bonding interactions with the substrate. The rate constants for association and dissociation of the peptide, measured by stopped-flow spectrometry, remain unchanged by the unnatural amino acid. The 5-FTrp-substituted enzyme exhibits a kcat of 73 s-1 as compared to 18 s-1 for the native enzyme toward 1-chloro-2,4-dinitrobenzene. That the increase in the turnover number is due to an enhanced rate of product release in the mutant is confirmed by the kinetics of the approach to equilibrium for binding of the product. The crystal structure of the 5-FTrp-containing enzyme was solved at a resolution of 2.0 A by difference Fourier techniques. The structure reveals local conformational changes in the structural elements that define the approach to the active site which are attributed to steric interactions of the fluorine atoms associated with 5-FTrp146 and 5-FTrp214 in domain II. These changes appear to result in the enhanced rate of product release. This structure represents the first of a protein substituted with 5-fluorotryptophan.     

Stereochemistry of an alpha, beta-elimination reaction by D-glucosaminate dehydratase

The proton NMR analysis of D-glucosaminate dehydratase reaction in D2O revealed the incorporation of a deuterium atom at C-3 carbon of the product, 2-keto-3-deoxy-D-gluconate. Based on the chemical shift of C-3 proton of the product and the coupling constant characteristic for the C-3 and C-4 axial-axial coupling in the 2C5 pyranose conformation, the deuterium is in the pro-S position. Thus, the dehydration of D-glucosaminate by the enzyme proceeds in a retention mode at C-3 carbon. Kinetic parameters show that the rate-determining step is the abstraction of alpha-proton from the substrate.     

Autocatalytic formation of a hydroxy group at C beta of trp171 in lignin peroxidase

In the high-resolution crystal structures of two lignin peroxidase isozymes from the white rot fungus Phanerochaete chrysosporium a significant electron density at single bond distance from the C beta of Trp171 was observed and interpreted as a hydroxy group. To further clarify the nature of this feature, we carried out tryptic digestion of the enzyme and isolated the Trp171 containing peptide. Under ambient conditions, this peptide shows an absorbance spectrum typical of tryptophan. At elevated temperature, however, the formation of an unusual absorbance spectrum with lambda max = 333 nm can be followed that is identical to that of N-acetyl-alpha, beta-didehydrotryptophanamide, resulting upon water elimination from beta-hydroxy tryptophan. The Trp171 containing tryptic peptide isolated from the recombinant and refolded lignin peroxidase produced from Escherichia coli does not contain the characteristic 333 nm absorbance band at any temperature. However, treatment with 3 equiv of H2O2 leads to complete hydroxylation of Trp171. Reducing substrates compete with this process, e.g., in the presence of 0.5 mM veratryl alcohol, about 7 equiv of H2O2 is necessary for complete modification. We conclude that the hydroxylation at the C beta of Trp171 is an autocatalytic reaction which occurs readily under conditions of natural turnover, e.g., in the ligninolytic cultures of P. chrysosporium, which are known to contain an oxidase-based H2O2-generating system. No dependence on dioxygen was found for this oxidative process. Chemical modification of fungal lignin peroxidase with the tryptophan-specific agent N-bromo succinimide leads to a drastically reduced activity with respect to the substrate veratryl alcohol. This suggests that Trp171 is involved in catalysis and that electron transfer from this surface residue to the oxidized heme cofactor is possible under steady-state conditions.     

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.     

Reactions of bovine liver catalase with superoxide radicals and hydrogen peroxide

The oxidized intermediates generated upon exposure of bovine liver catalase to hydrogen peroxide (H2O2) and superoxide radical (O2-) fluxes were examined with UV-visible spectrophotometry. H2O2 and O2- were generated by means of glucose/glucose oxidase and xanthine/xanthine oxidase systems. Serial overlay of absorption spectra in the Soret (350-450 nm) and visible (450-700 nm) regions showed that three oxidized intermediates, namely Compounds I, II and III, can be observed upon exposure of catalase to enzymatically generated H2O2 and O2-. Compound I is formed during the reaction of native enzyme with H2O2 and disappears in two ways: (i) via the catalytic reaction with H2O2 to restore native catalase and (ii) via the reaction with O2- to form Compound II. At low H2O2 concentrations (< 4.8 x 10(-9) M H2O2), Compound II reverts towards the native state mainly in a direct one-step reaction, whereas at higher H2O2 concentrations the pathway of Compound II back to the native enzyme involves Compound III. Formation of the latter from Compound II and H2O2 is irreversible and the rate constant of this reaction is 6.1 +/- 0.2 x 10(4) M-1 s-1. The formation of Compound III through the direct reaction of O2- with native enzyme has also been observed. Depending on the experimental conditions, the inactivation of catalase by O2- can be due to accumulation of Compound II ("slow" inhibition) or to the formation of Compound III ("rapid" inhibition) part of which leads to a dead end product. Formation of Compound III and of this dead end product are responsible for the irreversible inactivation in presence of an excess of H2O2.