Modulating electron density in the bound product, 4-hydroxybenzoyl-CoA, by mutations in 4-chlorobenzoyl-CoA dehalogenase near the 4-hydroxy group

The enzyme 4-chlorobenzoyl-CoA dehalogenase hydrolyzes 4-chlorobenzoyl-CoA (4-CBA-CoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA). Biochemical and crystallographic studies have identified a critical role for the dehalogenase residue Asp 145 in close proximity to the ligand's 4-hydroxy group in the structure of the product-enzyme complex. In the present study the effects of site selective mutations at Asp 145 on the product complex are explored by Raman spectroscopy. The spectral signatures of the WT-product complex, the large red shift in lambdamax, and the complete reorganization of the benzoyl ring modes in Raman data are absent for the D145E complex. The major spectral perturbations in the WT complex are brought about by strong electron "pull" at the benzoyl carbonyl and electron "push" by the side chain of Asp 145 near the 4-OH group. Acting in concert, these factors polarize the benzoyl's pi-electrons. Since the Raman data show that very strong electron pull occurs at the benzoyl's carbonyl in the D145E complex, it is apparent that the needed electron push near the benzoyl's 4-OH group is missing. Thus, very precise positioning of Asp 145's side chain near the benzoyl's 4-position is needed to bring about the dramatic electron reorganization seen in the WT complex, and this criterion cannot be met by the glutamate side chain with its additional CH2 group. For two other Asp145 mutants D145A and D145S that lack catalytic activity, Raman difference spectroscopic data for product complexes demonstrate the presence of a population of ionized product (i.e., 4-O-) in the active sites. The presence of the ionized phenolate form explains the observation that these complexes have highly red-shifted absorbance maxima with lambdamaxs near 400 nm. For the WT complex only the 4-OH form is seen, ionization being energetically expensive with the presence of the proximal negative charge on the Asp 145 side chain. Semiquantitative estimates of the pKa for the bound product in D145S and D145A indicate that this ionization lies in the pH 6.5-7.0 range. This is approximately 2 pH units below the pKa for the free product. The Raman spectrum of 4-dimethylaminobenzoyl-CoA undergoes major changes upon binding to dehalogenase. The bound form has two features near 1562 and 1529 cm-1 and therefore closely resembles the spectrum of product bound to wild-type enzyme, which underlines the quinonoid nature in these complexes. The use of a newly developed Raman system allowed us to obtain normal (nonresonance) Raman data for the dehalogenase complexes in the 100-300 microM range and heralds an important advance in the application of Raman spectroscopy to dilute solutions of macromolecules.     

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.     

Haloalkane dehalogenases: structure of a Rhodococcus enzyme

The hydrolytic haloalkane dehalogenases are promising bioremediation and biocatalytic agents. Two general classes of dehalogenases have been reported from Xanthobacter and Rhodococcus. While these enzymes share 30% amino acid sequence identity, they have significantly different substrate specificities and halide-binding properties. We report the 1.5 A resolution crystal structure of the Rhodococcus dehalogenase at pH 5.5, pH 7.0, and pH 5.5 in the presence of NaI. The Rhodococcus and Xanthobacter enzymes have significant structural homology in the alpha/beta hydrolase core, but differ considerably in the cap domain. Consistent with its broad specificity for primary, secondary, and cyclic haloalkanes, the Rhodococcus enzyme has a substantially larger active site cavity. Significantly, the Rhodococcus dehalogenase has a different catalytic triad topology than the Xanthobacter enzyme. In the Xanthobacter dehalogenase, the third carboxylate functionality in the triad is provided by D260, which is positioned on the loop between beta7 and the penultimate helix. The carboxylate functionality in the Rhodococcus catalytic triad is donated from E141. A model of the enzyme cocrystallized with sodium iodide shows two iodide binding sites; one that defines the normal substrate and product-binding site and a second within the active site region. In the substrate and product complexes, the halogen binds to the Xanthobacter enzyme via hydrogen bonds with the N(eta)H of both W125 and W175. The Rhodococcusenzyme does not have a tryptophan analogous to W175. Instead, bound halide is stabilized with hydrogen bonds to the N(eta)H of W118 and to N(delta)H of N52. It appears that when cocrystallized with NaI the Rhodococcus enzyme has a rare stable S-I covalent bond to S(gamma) of C187.     

Binding of protoporphyrin IX and metal derivatives to the active site of wild-type mouse ferrochelatase at low porphyrin-to-protein ratios

Resonance Raman (RR) spectroscopy is used to examine porphyrin substrate, product, and inhibitor interactions with the active site of murine ferrochelatase (EC 4.99.1.1), the terminal enzyme in the biosynthesis of heme. The enzyme catalyzes in vivo Fe(2+) chelation into protoporphyrin IX to give heme. The RR spectra of native ferrochelatase show that the protein, as isolated, contains varying amounts of endogenously bound high- or low-spin ferric heme, always at much less than 1 equiv. RR data on the binding of free-base protoporphyrin IX and its metalated complexes (Fe(III), Fe(II), and Ni(II)) to active wild-type protein were obtained at varying ratios of porphyrin to protein. The binding of ferric heme, a known inhibitor of the enzyme, leads to the formation of a low-spin six-coordinate adduct. Ferrous heme, the enzyme's natural product, binds in the ferrous high-spin five-coordinate state. Ni(II) protoporphyrin, a metalloporphyrin that has a low tendency toward axial ligation, becomes distorted when bound to ferrochelatase. Similarly for free-base protoporphyrin, the natural substrate of ferrochelatase, the RR spectra of porphyrin-protein complexes reveal a saddling distortion of the porphyrin. These results corroborate and extend our previous findings that porphyrin distortion, a crucial step of the catalytic mechanism, occurs even in the absence of bound metal substrate. Moreover, RR data reveal the presence of an amino acid residue in the active site of ferrochelatase which is capable of specific axial ligation to metals.     

Stable copper-nitrosyl formation by nitrite reductase in either oxidation state

Nitrite reductase (NiR) is an enzyme that uses type 1 and type 2 copper sites to reduce nitrite to nitric oxide during bacterial denitrification. A copper-nitrosyl intermediate is a proposed, yet poorly characterized feature of the NiR catalytic cycle. This intermediate is formally described as Cu(I)-NO+ and is proposed to be formed at the type 2 copper site after nitrite binding and electron transfer from the type 1 copper site. In this study, copper-nitrosyl complexes were formed by prolonged exposure of exogenous NO to crystals of wild-type and two variant forms of NiR from Alcaligenes faecalis (AfNiR), and the structures were determined to 1.8 A or better resolution. Exposing oxidized wild-type crystals to NO results in the reverse reaction and formation of nitrite that remains bound at the active site. In a type 1 copper site mutant (H145A) that is incapable of electron transfer to the type 2 site, the reverse reaction is not observed. Instead, in both oxidized and reduced H145A crystals, NO is observed bound in a side-on manner to the type 2 copper. In AfNiR, Asp98 forms hydrogen bonds to both substrate and product bound to the type 2 Cu. In the D98N variant, NO is bound side-on but is more disordered when observed for the wild-type enzyme. The solution EPR spectra of the crystallographically characterized NiR-NO complexes indicate the presence of an oxidized type 2 copper site and thus are interpreted as resulting from stable copper-nitrosyls and formally assigned as Cu(II)-NO-. A reaction scheme in which a second NO molecule is oxidized to nitrite can account for the formation of a Cu(II)-NO- species after exposure of the oxidized H145A variant to NO gas.     

Contributions of long-range electrostatic interactions to 4-chlorobenzoyl-CoA dehalogenase catalysis: a combined theoretical and experimental study

It is well established that electrostatic interactions play a vital role in enzyme catalysis. In this work, we report theory-guided mutation experiments that identified strong electrostatic contributions of a remote residue, namely, Glu232 located on the adjacent subunit, to 4-chlorobenzoyl-CoA dehalogenase catalysis. The Glu232Asp mutant was found to bind the substrate analogue 4-methylbenzoyl-CoA more tightly than does the wild-type dehalogenase. In contrast, the kcat for 4-chlorobenzoyl-CoA conversion to product was reduced 10000-fold in the mutant. UV difference spectra measured for the respective enzyme-ligand complexes revealed an approximately 3-fold shift in the equilibrium of the two active site conformers away from that inducing strong pi-electron polarization in the ligand benzoyl ring. Increased substrate binding, decreased ring polarization, and decreased catalytic efficiency indicated that the repositioning of the point charge in the Glu232Asp mutant might affect the orientation of the Asp145 carboxylate with respect to the substrate aromatic ring. The time course for formation and reaction of the arylated enzyme intermediate during a single turnover was measured for wild-type and Glu232Asp mutant dehalogenases. The accumulation of arylated enzyme in the wild-type dehalogenase was not observed in the mutant. This indicates that the reduced turnover rate in the mutant is the result of a slow arylation of Asp145, owing to decreased efficiency in substrate nucleophilic attack by Asp145. To rationalize the experimental observations, a theoretical model is proposed, which computes the potential of mean force for the nucleophilic aromatic substitution step using a hybrid quantum mechanical/molecular mechanical method. To this end, the removal or reorientation of the side chain charge of residue 232, modeled respectively by the Glu232Gln and Glu232Asp mutants, is shown to increase the rate-limiting energy barrier. The calculated 23.1 kcal/mol free energy barrier for formation of the Meisenheimer intermediate in the Glu232Asp mutant represents an increase of 6 kcal/mol relative to that of the wild-type enzyme, consistent with the 5.6 kcal/mol increase calculated from the difference in experimentally determined rate constants. On the basis of the combination of the experimental and theoretical evidence, we hypothesize that the Glu232(B) residue contributes to catalysis by providing an electrostatic force that acts on the Asp145 nucleophile.     

A resonance Raman study of substrate and inhibitor binding to protocatechuate-3,4-dioxygenase

Resonance Raman spectra were obtained for complexes of protocatechuate-3,4-dioxygenase with substrate and hydroxybenzoate inhibitors. The data establish metal coordination by these bound species and demonstrate further that tyrosine ligation, present in the resting enzyme, is not altered in the complexes. For the inhibitors, 3-chloro-4-hydroxybenzoate and 3-fluoro-4-hydroxybenzoate, the data are interpreted as indicating iron ligation by the phenolate functionality. For the substrate, 3,4-dihydroxyphenylproprionate, chelation via the o-dihydroxy grouping is proposed. In all three complexes tyrosine ligands present in the resting enzyme are not displaced. The inhibitor scattering intensity was utilized as an internal standard to estimate that two tyrosines are coordinated to the iron at the active site.     

Crystal structures of the substrate free-enzyme, and reaction intermediate of the HAD superfamily member, haloacid dehalogenase DehIVa from Burkholderia cepacia MBA4

DehIVa is a haloacid dehalogenase (EC 3.8.1.2) from the soil and water borne bacterium Burkholderia cepacia MBA4, which belongs to the functionally variable haloacid dehalogenase (HAD) superfamily of enzymes. The haloacid dehalogenases catalyse the removal of halides from haloacids resulting in a hydroxlated product. These enzymes are of interest for their potential to degrade recalcitrant halogenated environmental pollutants and their use in the synthesis of industrial chemicals. The haloacid dehalogenases utilise a nucleophilic attack on the substrate by an aspartic acid residue to form an enzyme-substrate ester bond and concomitantly cleaving of the carbon-halide bond and release of a hydroxylated product following ester hydrolysis. We present the crystal structures of both the substrate-free DehIVa refined to 1.93 A resolution and DehIVa covalently bound to l-2-monochloropropanoate trapped as a reaction intermediate, refined to 2.7 A resolution. Electron density consistent with a previously unidentified yet anticipated water molecule in the active site poised to donate its hydroxyl group to the product and its proton to the catalytic Asp11 is evident. It has been unclear how substrate enters the active site of this and related enzymes. The results of normal mode analysis (NMA) are presented and suggest a means whereby the predicted global dynamics of the enzyme allow for entry of the substrate into the active site. In the context of these results, the possible role of Arg42 and Asn178 in a "lock down" mechanism affecting active site access is discussed. In silico substrate docking of enantiomeric substrates has been examined in order to evaluate the enzymes enantioselectivity.     

The strength of dehalogenase-substrate hydrogen bonding correlates with the rate of Meisenheimer intermediate formation

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 aspartate as the nucleophile. Formation of the corresponding Meisenheimer complex (EMc) is followed by chloride ion expulsion which forms the arylated intermediate (EAr). This is then hydrolyzed to the product. In this paper, we explore the relationship between active site polarizing forces acting on the benzoyl carbonyl and the rate of formation of the Meisenheimer complex. The polarizing forces at the C[double bond]O group were modulated by introducing site-selected mutations (A112V, Y65D, G113A, G113S, G113N, and F64P), near the C[double bond]O binding site. Using either the substrate, 4-CBA-CoA, or the substrate analogue, 4-methylbenzoyl-CoA (4-MBA-CoA), Raman difference spectroscopy provided the position of the C[double bond]O stretching frequency (nu(C)[double bond](O)) for a total of 10 enzyme-ligand complexes. In turn, the values of the C[double bond]O frequencies could be converted to differences in effective hydrogen bonding strengths between members of the series, based on earlier model studies [Clarkson, J., Tonge, P. J., Taylor, K. L., Dunaway-Mariano, D., and Carey, P. (1997) Biochemistry 36, 10192-10199]. Catalysis in the F64P, G113A, G113S, and G113N dehalogenase mutants was very slow with k(cat) values ranging from 8 x 10(-3) to 7.6 x 10(-6) s(-1). The EAr intermediate did not accumulate to a detectable level on these enzymes during a single turnover. Catalysis in the Y65D and A112V dehalogenase mutants were almost as efficient as catalysis in wild-type dehalogenase with k(cat) values of 0.1-0.6 s(-1). In wild-type dehalogenase, 22% of the bound substrate accumulated as the EAr intermediate during a single turnover (k(obs) for EAr formation = 24 s(-(1)); in the Y65D mutant, the level of accumulation is 17% (k(obs) for EAr formation = 3 s(-1)), and in the A112V mutant, the level is 23% (k(obs) for EAr formation = 17 s(-1)). The k(obs) for EAr formation in wild-type dehalogenase and the more active dehalogenase mutants (Y65D and A112V) was taken to be an estimate of the k for EMc formation, and the k(obs) for EP formation in a single turnover was taken to be an estimate of the k for EMc formation in the severely impaired mutants (F64P, G113A, G113S, and G113N). A plot of the log k(obs) for EMc formation versus the C[double bond]O stretching frequency of bound 4-CBA-CoA (or 4-MBA-CoA) is a straight line (R(2) = 0.9584). Throughout the series, nu(C)[double bond](O) varied by 61 cm(-1), corresponding to the change in hydrogen bonding enthalpy of 67 kJ/mol. The results show that changes in polarizing forces at the benzoyl carbonyl are transmitted to the benzoyl (4) position and correlate with the rate of aromatic nucleophilic addition five chemical bonds away. Interestingly, the relationship between effective polarizing forces and reactivity seen here for dehalogenase is similar to that reported for the addition-elimination reaction involving the hydrolysis of a series of acyl serine proteases.     

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.     

17O-water and cyanide ligation by the active site iron of protocatechuate 3,4-dioxygenase. Evidence for displaceable ligands in the native enzyme and in complexes with inhibitors or transition state analogs

Hyperfine broadening is observable in the EPR spectrum of Brevibacterium fuscum protocatechuate 3,4-dioxygenase after lyophilization and rehydration in 17O-enriched water, demonstrating H2O ligation to the active site iron. Lack of detectable broadening in the sharp features of the spectra of three substrate complexes suggests that H2O is displaced by substrate. Water is bound in the monodentate complex with the competitive inhibitor 3-hydroxybenzoate which binds directly to the iron showing that two iron ligation sites can be occupied by nonprotein ligands. Ketonized substrate analogs which mimic a proposed transition state of the reaction cycle, 2-hydroxyisonicotinic acid N-oxide (2-OHINO) and 6-hydroxynicotinic acid N-oxide (6-OH NNO), have H2O bound in their final, bleached enzyme complexes, suggesting that these complexes are also monodentate. In contrast, a transient, initial complex of 6-OH NNO which is spectrally similar to the substrate complex, apparently does not have H2O bound. Cyanide binding occurs in two steps. The active site Fe3+ of the initial, rapidly formed, violet complex is high spin while that of the second, slowly formed, green complex is low spin; a unique state for mononuclear non-heme iron enzymes. The data suggest that the Fe-CN- and Fe-(CN-)2 complexes form sequentially. CN- binds to enzyme complexes with 2-OH INO and 6-OH NNO in one step to yield high spin Fe3+ species. In contrast, preformed substrate complexes prevent CN- binding. CN- binding eliminates the broadening due to 17O-water in the EPR spectra of both native enzyme and the enzyme-ketonized analog complexes. A model is proposed in which H2O is displaced by bidentate binding of the substrate but can potentially rebind after a subsequent substrate ketonization. The proximity of the vacatable H2O-binding site of the iron to the site of oxygen insertion suggests, however, that this site may serve to stabilize an oxygenated intermediate during the reaction cycle.     

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.     

Biochemical and structural characterisation of a haloalkane dehalogenase from a marine Rhodobacteraceae

A putative haloalkane dehalogenase has been identified in a marine Rhodobacteraceae and subsequently cloned and over-expressed in Escherichia coli. The enzyme has highest activity towards the substrates 1,6-dichlorohexane, 1-bromooctane, 1,3-dibromopropane and 1-bromohexane. The crystal structures of the enzyme in the native and product bound forms reveal a large hydrophobic active site cavity. A deeper substrate binding pocket defines the enzyme preference towards substrates with longer carbon chains. Arg136 at the bottom of the substrate pocket is positioned to bind the distal halogen group of extended di-halogenated substrates.     

Chromophoric cinnamic acid substrates as resonance Raman probes of the active site environment in native and unfolded alpha-chymotrypsin

Chromophoric [4-(dimethylamino)cinnamoyl]imidazole reacts with the serine protease alpha-chymotrypsin to form an acyl enzyme. At pHs below 4.0, the acyl enzyme turns over very slowly to yield the free acid. During this slow deacylation it is possible to obtain a very good resonance Raman spectrum of the acyl intermediate by using the 350.7-nm line of the krypton laser. The resonance Raman carbonyl frequency of the covalently bonded substrate and its wavelength at maximum intensity in the absorption spectrum of the acyl enzyme have been taken and used to monitor the active site environment. A comparison has been made of the absorption and Raman spectra of the acyl enzyme and those of the corresponding chromophoric methyl ester, aldehyde, and imidazole model compounds. A linear correlation is found between the wavelength of maximum absorption and the Raman frequency of the carbonyl group over a wide range of solvent conditions for each of the model compounds. By combining the Raman carbonyl frequency with the absorption maximum, we can determine that the bond order changes in the carbonyl bond of the bound substrate are not due to changes in the solvent, since the carbonyl frequency and the absorption maximum of the acyl enzyme do not fall on any of the linear correlations for the model compounds. The unusual spectroscopic properties of the bound substrate appear to be due to some specific enzyme-induced change in the substrate when it is bound at the active site. Thermal unfolding of the acyl enzymes changes both the carbonyl frequency of the acyl enzyme and its absorption maximum to completely different values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular mode of interaction of plant amine oxidase with the mechanism-based inhibitor 2-butyne-1,4-diamine.

2-Butyne-1,4-diamine (DABI) is a mechanism-based inhibitor of copper-containing plant amine oxidases; the number of turnovers that leads to enzyme inactivation is approximately 20. The product of DABI oxidation is a very reactive aminoallene that reacts with an essential nucleophilic group at the enzyme active site, forming a covalently bound pyrrole and producing an inactive enzyme. The inactivated enzyme shows a new absorption maximum at 295 nm and gives coloured derivatives with p-dimethylaminobenzaldehyde and p-dimethylaminocinnamaldehyde that are spectrally similar to the products of pyrrole treated with the above reagents. Resonance Raman spectra of the p-dimethylaminobenzaldehyde adduct of pyrrole and the inactivated enzyme show very high degree of similarity, supporting the idea that the product of inactivation is indeed a bound pyrrole. The bound pyrrole is formed already in the anaerobic step of the reaction, while the topa semiquinone radical is not affected, as shown by the EPR and stopped-flow absorption measurements. Peptides containing the DABI binding site were obtained by proteolysis of inactivated enzyme, isolated by HPLC and analysed by amino acid sequencing and MS. The crystal structure of the amine oxidase from pea has been determined; inhibition is caused mainly by the highly reactive DABI product, 4-amino-2-butynal, binding to a nucleophilic residue at the entrance to the substrate channel. As other DABI labelled peptides were also found and no free DABI product was detected by MS after complete inhibition of the enzyme, it is likely that the DABI product binds also to other solvent exposed nucleophilic residues on the enzyme surface.     

Substrate binding induces a cooperative conformational change in the 12S subunit of transcarboxylase: Raman crystallographic evidence

The 12S subunit of transcarboxylase is a 338 000 Da hexamer that transfers carboxlylate from methylmalonyl-CoA (MM-CoA) to biotin; in turn, the biotin transfers the carboxylate to pyruvate on another subunit, the 5S. Here, Raman difference microscopy is used to study the binding of substrate and product, and their analogues, to single crystals of 12S. A single crystal is the medium of choice because it provides Raman data of unprecedented quality. Crystalline ligand-protein complexes were formed by cocrystallization or by the soaking in/soaking out method. Raman difference spectra were obtained by subtracting the spectrum of the apo crystal from that of a crystal with the substrate or product bound. Raman difference spectra from crystals with the substrate bound are dominated by bands from the protein's amide bonds and aromatic side chain residues. In contrast, Raman difference spectra involving the product, propionyl-CoA, are dominated by modes from the ligand. These results show that substrate binding triggers a conformational change in 12S, whereas product binding does not. The conformational change involves an increase in the amount of alpha-helix since markers for this secondary structure are prominent in the difference spectra of the substrate complex. The number of MM-CoA ligands bound per 12S hexamer can be gauged from the intensity of the MM-CoA Raman features and the fact that the protein concentration in the crystals is known from X-ray crystallographic data. Most crystal samples had six MM-CoAs per hexamer although a few, from different soaking experiments, contained only 1-2. However, both sets of crystals showed the same degree of protein conformational change, indicating that the change induced by the substrate is cooperative. This effect allowed us to record the Raman spectrum of bound MM-CoA without interference from protein modes; the Raman spectrum of a 12S crystal containing 2 MM-CoA ligands per hexamer was subtracted from the Raman spectrum of a 12S crystal containing six MM-CoA ligands per hexamer. The conformational change is reversible and can be controlled by soaking out or soaking in the ligand, using either concentrated ammonium sulfate solutions or the solution used in the crystallization trials. Malonyl-CoA also binds to 12S crystals and brings about conformational changes identical to those seen for MM-CoA; in addition, butyryl-CoA binds and behaves in a manner similar to propionyl-CoA. These data implicate the -COO- group on MM-CoA (that is transferred to biotin in the reaction on the intact enzyme) as the agent bringing about the cooperative conformational change in 12S.     

Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle

Escherichia coli dihydrofolate reductase (DHFR) has several flexible loops surrounding the active site that play a functional role in substrate and cofactor binding and in catalysis. We have used heteronuclear NMR methods to probe the loop conformations in solution in complexes of DHFR formed during the catalytic cycle. To facilitate the NMR analysis, the enzyme was labeled selectively with [(15)N]alanine. The 13 alanine resonances provide a fingerprint of the protein structure and report on the active site loop conformations and binding of substrate, product, and cofactor. Spectra were recorded for binary and ternary complexes of wild-type DHFR bound to the substrate dihydrofolate (DHF), the product tetrahydrofolate (THF), the pseudosubstrate folate, reduced and oxidized NADPH cofactor, and the inactive cofactor analogue 5,6-dihydroNADPH. The data show that DHFR exists in solution in two dominant conformational states, with the active site loops adopting conformations that closely approximate the occluded or closed conformations identified in earlier X-ray crystallographic analyses. A minor population of a third conformer of unknown structure was observed for the apoenzyme and for the disordered binary complex with 5,6-dihydroNADPH. The reactive Michaelis complex, with both DHF and NADPH bound to the enzyme, could not be studied directly but was modeled by the ternary folate:NADP(+) and dihydrofolate:NADP(+) complexes. From the NMR data, we are able to characterize the active site loop conformation and the occupancy of the substrate and cofactor binding sites in all intermediates formed in the extended catalytic cycle. In the dominant kinetic pathway under steady-state conditions, only the holoenzyme (the binary NADPH complex) and the Michaelis complex adopt the closed loop conformation, and all product complexes are occluded. The catalytic cycle thus involves obligatory conformational transitions between the closed and occluded states. Parallel studies on the catalytically impaired G121V mutant DHFR show that formation of the closed state, in which the nicotinamide ring of the cofactor is inserted into the active site, is energetically disfavored. The G121V mutation, at a position distant from the active site, interferes with coupled loop movements and appears to impair catalysis by destabilizing the closed Michaelis complex and introducing an extra step into the kinetic pathway.     

Resonance Raman evidence for substrate reorginization in the active site of papain.

Resonance Raman spectra were obtained for the acylenzyme 4-dimethylamino-3-nitro(alpha-benzamido)cinnamoyl-papain prepared using the chromophoric substrate methyl 4-dimethylamino-3-nitro(alpha-benzamido)cinnamate. These spectra contained vibrational spectral data of the acyl residue while covalently attached to the active site and could be used to follow directly acylation and deacylation kinetics. Spectra were obtained at pH values ranging from those where the acyl-enzyme is relatively stable (pH 3.0, tau 1/2 congruent to 800 s) to those where it is relatively unstable (pH 9.2, tau 1/2 congruent to 223 s). Throughout this range acyl-enzyme spectra differed completely from that of the free substrate or the product (4-dimethylamino-3-nitro(alpha-benzamido)cinnamic acid) indicating that a structural change occurred on combination with the active site. The spectra are consistent with rearrangement of the alpha-benzamido group in the bound substrate, -NH--C(==O)Ph becoming --N==C(--OX)Ph, where the bonding to oxygen is unknown. Superimposed on these large differences, small changes in acyl-enzyme spectra also occurred as pH was raised to decrease the half-life. All of the above spectral perturbations are consistent with a structural change in the acyl-enzyme which precedes the rate-determining step in deacylation. Thus, deacylation proceeds from an acyl residue structure differing from that of the substrate in solution. Upon acid denaturation the spectrum characteristic of the intermediate reverts to one closely resembling the substrate, demonstrating that a functioning active site is necessary to produce the observed differences. Spectra in D2O of native acyl-enzyme were identical with those in H2O, indicating that the observed differences in rate constant were not due to solvent-induced structural changes. Activated papain purified by crystallization or by affinity chromatography formed the acyl-enzyme. However, the kinetics of formation and deacylation differed between these materials, as did the spectral properties. Small differences in active-site structure are considered to be responsible for this effect, and it is suggested that such spectral perturbations may be useful in directly relating small differences in structure of the substrate in the active site with corresponding differences in kinetics.     

Crystal structure of tobacco etch virus protease shows the protein C terminus bound within the active site

Tobacco etch virus (TEV) protease is a cysteine protease exhibiting stringent sequence specificity. The enzyme is widely used in biotechnology for the removal of the affinity tags from recombinant fusion proteins. Crystal structures of two TEV protease mutants as complexes with a substrate and a product peptide provided the first insight into the mechanism of substrate specificity of this enzyme. We now report a 2.7A crystal structure of a full-length inactive C151A mutant protein crystallised in the absence of peptide. The structure reveals the C terminus of the protease bound to the active site. In addition, we determined dissociation constants of TEV protease substrate and product peptides using isothermal titration calorimetry for various forms of this enzyme. Data suggest that TEV protease could be inhibited by the peptide product of autolysis. Separate modes of recognition for native substrates and the site of TEV protease self-cleavage are proposed.     

Purification and properties of a high-affinity L-2-haloacid dehalogenase from Azotabacter sp. strain RC26

A monomeric 29 kDa protein showing dehalogenase activity on several halogenated carboxylic acids has been purified from Azotobacter sp. strain RC26. The purified enzyme is specific for the L isomer of optically active 2-haloacids leading to the inversion of the product configuration. The dehalogenase is active at temperatures ranging from 30 to 60C and shows a relatively high affinity for the substrate. The combined thermal stability, high substrate affinity and resistance to enzyme inhibitors found for the RC26 dehalogenase may be relevant for its use as catalyst in biotransformation processes.