Mechanism of the severe inhibition of tetrachlorohydroquinone dehalogenase by its aromatic substrates

Tetrachlorohydroquinone (TCHQ) dehalogenase catalyzes the conversion of TCHQ to 2,6-dichlorohydroquinone during degradation of pentachlorophenol by Sphingobium chlorophenolicum. TCHQ dehalogenase is a member of the glutathione S-transferase superfamily. Members of this superfamily typically catalyze nucleophilic attack of glutathione upon an electrophilic substrate to form a glutathione conjugate and contain a single glutathione binding site in each monomer of the typically dimeric enzyme. TCHQ dehalogenase, in contrast to most members of the superfamily, is a monomer and uses 2 equiv of glutathione to catalyze a more complex reaction. The first glutathione is involved in formation of a glutathione conjugate, while the second is involved in the final step of the reaction, a thiol-disulfide exchange reaction that regenerates the free enzyme and forms GSSG. TCHQ dehalogenase is severely inhibited by its aromatic substrates, TCHQ and trichlorohydroquinone (TriCHQ). TriCHQ acts as a noncompetitive inhibitor of the thiol-disulfide exchange reaction required to regenerate the free form of the enzyme. In addition, dissociation of the GSSG product is inhibited by TriCHQ. The thiol-disulfide exchange reaction is the rate-limiting step in the reductive dehalogenation reaction under physiological conditions.     

A trade-off between catalytic power and substrate inhibition in TCHQ dehalogenase

Tetrachlorohydroquinone (TCHQ) dehalogenase is profoundly inhibited by its aromatic substrates, TCHQ and trichlorohydroquinone (TriCHQ). Surprisingly, mutations that change Ile12 to either Ser or Ala give an enzyme that shows no substrate inhibition. We have previously shown that TriCHQ is a noncompetitive inhibitor of the thiol-disulfide exchange reaction between glutathione and ESSG, a covalent adduct between Cys13 and glutathione formed during dehalogenation of the substrate. Substrate inhibition of the thiol-disulfide exchange reaction is less severe in the I12S and I12A mutant enzymes, primarily due to weaker binding of TriCHQ to ESSG. These mutations also result in a decrease in the rate of dehalogenation. Because the rate-limiting step in the I12S and I12A enzymes is dehalogenation, rather than the thiol-disulfide exchange reaction, the relatively modest inhibition of the thiol-disulfide exchange reaction does not affect the overall rate of turnover.     

Pre-steady-state kinetic studies of the reductive dehalogenation catalyzed by tetrachlorohydroquinone dehalogenase

Tetrachlorohydroquinone dehalogenase catalyzes two successive reductive dehalogenation reactions in the pathway for degradation of pentachlorophenol in the soil bacterium Sphingobium chlorophenolicum. We have used pre-steady-state kinetic methods to probe both the mechanism and the rates of elementary steps in the initial stages of the reductive dehalogenation reaction. Binding of trichlorohydroquinone (TriCHQ) to the active site is followed by rapid deprotonation to form TriCHQ-2 and subsequent formation of 3,5,6-trichloro-4-hydroxycyclohexa-2,4-dienone (TriCHQ*). Further conversion of TriCHQ* to 2,6-dichlorohydroquinone (DCHQ) proceeds only in the presence of glutathione. Conversion of TriCHQ to DCHQ during the first turnover is quite rapid, occurring at about 25 s-1 when the enzyme is saturated with TriCHQ and glutathione. The rate of subsequent turnovers is limited by the rate of the thiol-disulfide exchange reaction required to regenerate the free enzyme after turnover, a reaction that is intrinsically less difficult, but is hampered by premature binding of the aromatic substrate to the active site before the catalytic cycle is completed.     

Recruitment of a double bond isomerase to serve as a reductive dehalogenase during biodegradation of pentachlorophenol

Tetrachlorohydroquinone dehalogenase catalyzes the replacement of chlorine atoms on tetrachlorohydroquinone and trichlorohydroquinone with hydrogen atoms during the biodegradation of pentachlorophenol by Sphingomonas chlorophenolica. The sequence of the active site region of tetrachlorohydroquinone dehalogenase is very similar to those of the corresponding regions of maleylacetoacetate isomerases, enzymes that catalyze the glutathione-dependent isomerization of a cis double bond in maleylacetoacetate to the trans configuration during the catabolism of phenylalanine and tyrosine. Furthermore, tetrachlorohydroquinone dehalogenase catalyzes the isomerization of maleylacetone (an analogue of maleylacetoacetate) at a rate nearly comparable to that of a bona fide bacterial maleylacetoacetate isomerase. Since maleylacetoacetate isomerase is involved in a common and presumably ancient pathway for catabolism of tyrosine, while tetrachlorohydroquinone dehalogenase catalyzes a more specialized reaction, it is likely that tetrachlorohydroquinone dehalogenase arose from a maleylacetoacetate isomerase. The substrates and overall transformations involved in the dehalogenation and isomerization reactions are strikingly different. This enzyme provides a remarkable example of Nature's ability to recruit an enzyme with a useful structural scaffold and elaborate upon its basic catalytic capabilities to generate a catalyst for a newly needed reaction.     

A new -2-haloacid dehalogenase acting on 2-haloacid amides: purification, characterization, and mechanism

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of - and -2-haloalkanoic acids to produce the corresponding - and -2-hydroxyalkanoic acids, respectively. We have constructed an overproduction system for -2-haloacid dehalogenase from Pseudomonas putida PP3 ( -DEX 312) and purified the enzyme to analyze the reaction mechanism. When a single turnover reaction of -DEX 312 was carried out in H₂¹⁸O by use of a large excess of the enzyme with - or -2-chloropropionate as a substrate, the lactate produced was labeled with ¹⁸O. This indicates that the solvent water molecule directly attacked the substrate and that its oxygen atom was incorporated into the product. This reaction mechanism contrasts with that of -2-haloacid dehalogenase, which has an active-site carboxylate group that attacks the substrate to displace the halogen atom. -DEX 312 resembles -2-haloacid dehalogenase from Pseudomonas sp. 113 ( -DEX 113) in that the reaction proceeds with a direct attack of a water molecule on the substrate. However, -DEX 312 is markedly different from -DEX 113 in its substrate specificity. We found that -DEX 312 catalyzes the hydrolytic dehalogenation of 2-chloropropionamide and 2-bromopropionamide, which do not serve as substrates for -DEX 113. -DEX 312 is the first enzyme that catalyzes the dehalogenation of 2-haloacid amides.     

The reaction catalyzed by tetrachlorohydroquinone dehalogenase does not involve nucleophilic aromatic substitution.

Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachlorohydroquinone and trichlorohydroquinone during the biodegradation of the xenobiotic compound pentachlorophenol by Sphingobium chlorophenolicum. The mechanism of this transformation is of interest because it is unusual and difficult, and because aerobic microorganisms rarely catalyze reductive dehalogenation reactions. Tetrachlorohydroquinone dehalogenase is a member of the glutathione S-transferase superfamily. Many enzymes in this superfamily are capable of catalyzing nucleophilic aromatic substitution reactions. On the basis of this precedent, we have considered a mechanism for tetrachlorohydroquinone dehalogenase that involves a nucleophilic aromatic substitution reaction, either via an S(N)Ar mechanism or an S(RN)1-like mechanism, in the initial part of the reaction. Mechanistic studies were carried out with the wild type enzyme and with the C13S mutant enzyme, which catalyzes only the initial steps in the reaction. Three findings eliminate the possibility of a nucleophilic aromatic substitution reaction. First, the product of such a reaction, 2,3,5-trichloro-6-S-glutathionylhydroquinone, is not a kinetically competent intermediate. Second, the enzyme can carry out the reaction when the substrate is deprotonated at the active site. Nucleophilic aromatic substitution should not be possible when the substrate is negatively charged. Third, substantial normal solvent kinetic isotope effects on k(cat) and k(cat)/K(M,TriCHQ) are observed. Nonenzymatic and enzymatic nucleophilic S(N)Ar reactions typically show inverse solvent kinetic isotope effects.     

Reactivity of asparagine residue at the active site of the D105N mutant of fluoroacetate dehalogenase from Moraxella sp. B

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon–fluorine bond of fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the α-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation.     

Crystal structure of maleylacetoacetate isomerase/glutathione transferase zeta reveals the molecular basis for its remarkable catalytic promiscuity

Maleylacetoacetate isomerase (MAAI), a key enzyme in the metabolic degradation of phenylalanine and tyrosine, catalyzes the glutathione-dependent isomerization of maleylacetoacetate to fumarylacetoacetate. Deficiencies in enzymes along the degradation pathway lead to serious diseases including phenylketonuria, alkaptonuria, and the fatal disease, hereditary tyrosinemia type I. The structure of MAAI might prove useful in the design of inhibitors that could be used in the clinical management of the latter disease. Here we report the crystal structure of human MAAI at 1.9 A resolution in complex with glutathione and a sulfate ion which mimics substrate binding. The enzyme has previously been shown to belong to the zeta class of the glutathione S-transferase (GST) superfamily based on limited sequence similarity. The structure of MAAI shows that it does adopt the GST canonical fold but with a number of functionally important differences. The structure provides insights into the molecular bases of the remarkable array of different reactions the enzyme is capable of performing including isomerization, oxygenation, dehalogenation, peroxidation, and transferase activity.     

Tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans: substrate specificity of the native enzyme and its corrinoid cofactor

The substrate specificity of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivoransand its corrinoid cofactor were studied. Besides reduced methyl viologen, titanium(III) citrate could serve as electron donor for reductive dehalogenation of tetrachloroethene (PCE) and trichloroethene to cis-1,2-dichloroethene. In addition to chlorinated ethenes, chlorinated propenes were reductively dechlorinated solely by the native enzyme. trans-1,3-Dichloropropene, 1,1,3-trichloropropene and 2,3-dichloropropene were reduced to a mixture of mono-chloropropenes, 1,1-dichloropropene, and 2-chloropropene, respectively. Other halogenated compounds that were rapidly reduced by the enzyme were also dehalogenated abiotically by the heat-inactivated enzyme and by commercially available cyanocobalamin. The rate of this abiotic reaction was dependent on the number and type of halogen substituents and on the type of catalyst. The corrinoid cofactor purified from the tetrachloroethene dehalogenase of D. multivorans exhibited an activity about 50-fold higher than that of cyanocobalamin (vitamin B(12)) with trichloroacetate as electron acceptor, indicating that the corrinoid cofactor of the PCE dehalogenase is not cyanocobalamin. Corrinoids catalyzed the rapid dehalogenation of trichloroacetic acid. The rate was proportional to the amount of, e.g. cyanocobalamin; therefore, the reductive dehalogenation assay can be used for the sensitive and rapid quantification of this cofactor.     

DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a new class of dehalogenase catalyzing hydrolytic dehalogenation not involving enzyme-substrate ester intermediate.

DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 (DL-DEX 113) catalyzes the hydrolytic dehalogenation of D- and L-2-haloalkanoic acids, producing the corresponding L- and D-2-hydroxyalkanoic acids, respectively. Every halidohydrolase studied so far (L-2-haloacid dehalogenase, haloalkane dehalogenase, and 4-chlorobenzoyl-CoA dehalogenase) has an active site carboxylate group that attacks the substrate carbon atom bound to the halogen atom, leading to the formation of an ester intermediate. This is subsequently hydrolyzed, resulting in the incorporation of an oxygen atom of the solvent water molecule into the carboxylate group of the enzyme. In the present study, we analyzed the reaction mechanism of DL-DEX 113. When a single turnover reaction of DL-DEX 113 was carried out with a large excess of the enzyme in H(2)(18)O with a 10 times smaller amount of the substrate, either D- or L-2-chloropropionate, the major product was found to be (18)O-labeled lactate by ionspray mass spectrometry. After a multiple turnover reaction in H(2)(18)O, the enzyme was digested with trypsin or lysyl endopeptidase, and the molecular masses of the peptide fragments were measured with an ionspray mass spectrometer. No peptide fragments contained (18)O. These results indicate that the H(2)(18)O of the solvent directly attacks the alpha-carbon of 2-haloalkanoic acid to displace the halogen atom. This is the first example of an enzymatic hydrolytic dehalogenation that proceeds without producing an ester intermediate.     

Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-haloacid dehalogenases.

DL-2-Haloacid dehalogenase from Pseudomonas sp. strain 113 (DL-DEX) catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids to produce the corresponding L- and D-2-hydroxyalkanoic acids, respectively, with inversion of the C2 configuration. DL-DEX is a unique enzyme: it acts on the chiral carbon of the substrate and uses both enantiomers as equivalent substrates. We have isolated and sequenced the gene encoding DL-DEX. The open reading frame consists of 921 bp corresponding to 307 amino acid residues. No sequence similarity between DL-DEX and L-2-haloacid dehalogenases was found. However, DL-DEX had significant sequence similarity with D-2-haloacid dehalogenase from Pseudomonas putida AJ1, which specifically acts on D-2-haloalkanoic acids: 23% of the total amino acid residues of DL-DEX are conserved. We mutated each of the 26 residues with charged and polar side chains, which are conserved between DL-DEX and D-2-haloacid dehalogenase. Thr65, Glu69, and Asp194 were found to be essential for dehalogenation of not only the D- but also the L-enantiomer of 2-haloalkanoic acids. Each of the mutant enzymes, whose activities were lower than that of the wild-type enzyme, acted on both enantiomers of 2-haloacids as equivalent substrates in the same manner as the wild-type enzyme. We also found that each enantiomer of 2-chloropropionate competitively inhibits the enzymatic dehalogenation of the other. These results suggest that DL-DEX has a single and common catalytic site for both enantiomers.     

pH Dependence of the Isomerase Activity of Protein Disulfide Isomerase: Insights into its Functional Relevance

The isomerase efficacy of the oxidoreductase, protein disulfide isomerase (PDI), has been examined by a simple method. Using this technique, the pH-dependence of relative efficiency of isomerization reactions by PDI has been evaluated and its impact on a key structure-forming step in the oxidative folding pathway of a model protein determined. Results reveal that PDI has a greater relative impact on thiol-disulfide reshuffling (isomerization) reactions and consequently the structure-forming step in oxidative folding at pH 7, as opposed to pH’s 8 and 9. These results suggest that PDI, which possesses an anomalously low thiol pKa, is fine-tuned to catalyze oxidative folding in the lumen of the endoplasmic reticulum where the ambient pH of ∼7 would otherwise retard thiol-disulfide exchange reactions and hinder acquisition of the native fold. The pH-dependent impact on isomerization catalysis has important implications for the development of synthetic chaperones for in vivo and in vitro applications.     

Purification and characterization of a novel 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of Desulfomonile tiedjei DCB-1.

Although reductive dehalogenation by anaerobic microorganisms offers great potential for the degradation of halocarbons, little is known about the biochemical mechanisms involved. It has previously been demonstrated that the dehalogenase activity involved in 3-chlorobenzoate dehalogenation by Desulfomonile tiedjei DCB-1 is present in the membrane fraction of the cell extracts. We report herein the purification of a 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of D. tiedjei DCB-1. The dehalogenase activity was monitored by the conversion of 3-chlorobenzoate to benzoate with reduced methyl viologen as a reducing agent. The membrane fraction of the cell extracts was obtained by ultracentrifugation, and the membrane proteins were solubilized with either the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) or Triton X-100 in the presence of glycerol. The solubilized dehalogenase was purified by ammonium sulfate fractionation and a combination of anion exchange, hydroxyapatite, and hydrophobic interaction chromatographies. This procedure yielded about 7% of the total dehalogenase activity with a 120-fold increase in specific activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified dehalogenase consisted of two subunits with molecular weights of 64,000 and 37,000. The enzyme converted 3-chlorobenzoate to benzoate at its highest specific activity in 10 mM potassium phosphate buffer (pH 7.2) at 38 degrees C. The enzyme was yellow and probably a heme protein. The enzyme had an adsorbance peak at 408 nm. The dithionite-reduced enzyme displayed absorbance peaks at 416, 522, and 550 nm. The dithionite-reduced enzyme was able to complex with carbon monoxide. The nature of the heme chromophore is currently unknown.     

Sulfhydryl oxidation, not disulfide isomerization, is the principal function of protein disulfide isomerase in yeast Saccharomyces cerevisiae

Protein disulfide isomerase (PDI) is an essential protein folding assistant of the eukaryotic endoplasmic reticulum that catalyzes both the formation of disulfides during protein folding (oxidase activity) and the isomerization of disulfides that may form incorrectly (isomerase activity). Catalysis of thiol-disulfide exchange by PDI is required for cell viability in Saccharomyces cerevisiae, but there has been some uncertainty as to whether the essential role of PDI in the cell is oxidase or isomerase. We have studied the ability of PDI constructs with high oxidase activity and very low isomerase activity to complement the chromosomal deletion of PDI1 in S. cerevisiae. A single catalytic domain of yeast PDI (PDIa') has 50% of the oxidase activity but only 5% of the isomerase activity of wild-type PDI in vitro. Titrating the expression of PDI using the inducible/repressible GAL1-10 promoter shows that the amount of wild-type PDI protein needed to sustain a normal growth rate is 60% or more of the amount normally expressed from the PDI1 chromosomal location. A single catalytic domain (PDIa') is needed in molar amounts that are approximately twice as high as those required for wild-type PDI, which contains two catalytic domains. This comparison suggests that high (>60%) PDI oxidase activity is critical to yeast growth and viability, whereas less than 6% of its isomerase activity is needed.     

GSTB1-1 from Proteus mirabilis: a snapshot of an enzyme in the evolutionary pathway from a redox enzyme to a conjugating enzyme

The native form of the bacterial glutathione transferase B1-1 (EC ) is characterized by one glutathione (GSH) molecule covalently linked to Cys-10. This peculiar disulfide, only found in the Beta and Omega class glutathione S-transferases (GSTs) but absent in all other GSTs, prompts questions about its role and how GSH can be activated and utilized in the reaction normally performed by GSTs. Stopped-flow and spectroscopic experiments suggest that, in the native enzyme (GSTB1-1ox), a second GSH molecule is present, albeit transiently, in the active site. This second GSH binds to the enzyme through a bimolecular interaction followed by a fast thiol-disulfide exchange with the covalently bound GSH. The apparent pK(a) of the non-covalently bound GSH is lowered from 9.0 to 6.4 +/- 0.2 in similar fashion to other GSTs. The reduced form of GSTB1-1 (GSTB1-1red) binds GSH 100-fold faster and also induces a more active deprotonation of the substrate with an apparent pK(a) of 5.2 +/- 0.1. Apparently, the absence of the mixed disulfide does not affect k(cat) and K(m) values in the GST conjugation activity, which is rate-limited by the chemical step both in GSTB1-1red and in GSTB1-1ox. However, GSTB1-1ox follows a steady-state random sequential mechanism whereas a rapid-equilibrium random sequential mechanism is adopted by GSTB1-1red. Remarkably, GSTB1-1ox and GSTB1-1red are equally able to catalyze a glutaredoxin-like catalysis using cysteine S-sulfate and hydroxyethyl disulfide as substrates. Cys-10 is an essential residue in this redox activity, and its replacement by alanine abolishes this enzymatic activity completely. It appears that GSTB1-1 behaves like an "intermediate enzyme" between the thiol-disulfide oxidoreductase and the GST superfamilies.     

Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site

Haloalcohol dehalogenases are bacterial enzymes that catalyze the cofactor-independent dehalogenation of vicinal haloalcohols such as the genotoxic environmental pollutant 1,3-dichloro-2-propanol, thereby producing an epoxide, a chloride ion and a proton. Here we present X-ray structures of the haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1, and complexes of the enzyme with an epoxide product and chloride ion, and with a bound haloalcohol substrate mimic. These structures support a catalytic mechanism in which Tyr145 of a Ser-Tyr-Arg catalytic triad deprotonates the haloalcohol hydroxyl function to generate an intramolecular nucleophile that substitutes the vicinal halogen. Haloalcohol dehalogenases are related to the widespread family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDR family), which use a similar Ser-Tyr-Lys/Arg catalytic triad to catalyze reductive or oxidative conversions of various secondary alcohols and ketones. Our results reveal the first structural details of an SDR-related enzyme that catalyzes a substitutive dehalogenation reaction rather than a redox reaction, in which a halide-binding site is found at the location of the NAD(P)H binding site. Structure-based sequence analysis reveals that the various haloalcohol dehalogenases have likely originated from at least two different NAD-binding SDR precursors.     

Evolution of a metabolic pathway for degradation of a toxic xenobiotic: the patchwork approach

The pathway for degradation of the xenobiotic pesticide pentachlorophenol in Sphingomonas chlorophenolica probably evolved in the past few decades by the recruitment of enzymes from two other catabolic pathways. The first and third enzymes in the pathway, pentachlorophenol hydroxylase and 2,6-dichlorohydroquinone dioxygenase, may have originated from enzymes in a pathway for degradation of a naturally occurring chlorinated phenol. The second enzyme, a reductive dehalogenase, may have evolved from a maleylacetoacetate isomerase normally involved in degradation of tyrosine. This apparently recently assembled pathway does not function very well: pentachlorophenol hydroxylase is quite slow, and tetrachlorohydroquinone dehalogenase is subject to severe substrate inhibition.     

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion

Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.     

Light modulation of enzyme activity: activation of the light effect mediators by reduction and modulation of enzyme activity by thiol-disulfide exchange

Light and dark modulation experiments with pea (Pisum sativum L.) chloroplast stromal fractions pretreated with dithiothreitol (to reduce protein disulfide bonds) or with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (to block sulfhydryl groups) suggest that light modulation involves thiol-disulfide exchange on the modulatable stromal enzyme protein. Light-dependent reduction of DTNB involves a photosynthetic electron transport chain component located on the reducing side of photosystem I prior to ferredoxin; DTNB may be acting as a light effect mediator substitute. The thylakoid-bound light effect mediator system, then, in its light-activated reduced form probably catalyzes thiol-disulfide exchange reactions on stromal enzymes.     

Raman evidence for Meisenheimer complex formation in the hydrolysis reactions of 4-fluorobenzoyl- and 4-nitrobenzoyl-coenzyme A catalyzed by 4-chlorobenzoyl-coenzyme A dehalogenase

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA to 4-hydroxybenzoyl-CoA by using an active site Asp145 carboxylate as the nucleophile. Formation of the corresponding Meisenheimer complex (EMc) is followed by chloride ion expulsion to form arylated enzyme (EAr). The EAr is then hydrolyzed to product. In this paper, we report the kinetics for dehalogenase-catalyzed 4-fluorobenzoyl-CoA (4-FBA-CoA) and 4-nitrobenzoyl-CoA (4-NBA-CoA) hydrolysis and provide Raman spectral evidence for the accumulation of EMc in these reactions. The 4-FBA-CoA and 4-NBA-CoA substrate analogues were selected for the poor leaving group ability of their C(4) substituents. Thus, the formation of the EAr from EMc should be hindered, giving rise to a quasi-steady-state equilibrium between EMc and the Michaelis complex. Detailed kinetic studies were carried out to quantitate the composition of the reaction mixtures. Quench experiments demonstrated that significant populations of EAr do not exist in reaction mixtures involving the 4-F- or 4-N-substrates. A kinetic model enabled us to estimate that approximately 10-20% of the enzyme-substrate complexes in the reaction mixtures are present as EMc. Raman difference spectra of 4-NBA-CoA and 4-FBA-CoA bound to WT and H90Q mutant dehalogenase have broad features near 1500 and 1220 cm(-1) that are absent in the free ligand. Crucially, these features are also absent in the Raman spectra of the complexes involving the D145A dehalogenase mutant that are unable to form an EMc. Quantum mechanical calculations, at the DFT level, provide strong support for assigning the novel 1500 and 1220 cm(-1) features to an EMc.