Transient Kinetic Studies Support Refinements to the Chemical and Kinetic Mechanisms of Aminolevulinate Synthase
2007; Elsevier BV; Volume: 282; Issue: 32 Linguagem: Inglês
10.1074/jbc.m609330200
ISSN1083-351X
AutoresGregory A. Hunter, Junshun Zhang, Glória C. Ferreira,
Tópico(s)HIV/AIDS drug development and treatment
Resumo5-Aminolevulinate synthase catalyzes the pyridoxal 5′-phosphate-dependent condensation of glycine and succinyl-CoA to produce carbon dioxide, CoA, and 5-aminolevulinate, in a reaction cycle involving the mechanistically unusual successive cleavage of two amino acid substrate α-carbon bonds. Single and multiple turnover rapid scanning stopped-flow experiments have been conducted from pH 6.8–9.2 and 5–35 °C, and the results, interpreted within the framework of the recently solved crystal structures, allow refined characterization of the central kinetic and chemical steps of the reaction cycle. Quinonoid intermediate formation occurs with an apparent pKa of 7.7 ± 0.1, which is assigned to His-207 acid-catalyzed decarboxylation of the α-amino-β-ketoadipate intermediate to form an enol that is in rapid equilibrium with the 5-aminolevulinate-bound quinonoid species. Quinonoid intermediate decay occurs in two kinetic steps, the first of which is acid-catalyzed with a pKa of 8.1 ± 0.1, and is assigned to protonation of the enol by Lys-313 to generate the product-bound external aldimine. The second step of quinonoid decay defines kcat and is relatively pH-independent and is assigned to opening of the active site loop to allow ALA dissociation. The data support important refinements to both the chemical and kinetic mechanisms and indicate that 5-aminolevulinate synthase operates under the stereoelectronic control predicted by Dunathan's hypothesis. 5-Aminolevulinate synthase catalyzes the pyridoxal 5′-phosphate-dependent condensation of glycine and succinyl-CoA to produce carbon dioxide, CoA, and 5-aminolevulinate, in a reaction cycle involving the mechanistically unusual successive cleavage of two amino acid substrate α-carbon bonds. Single and multiple turnover rapid scanning stopped-flow experiments have been conducted from pH 6.8–9.2 and 5–35 °C, and the results, interpreted within the framework of the recently solved crystal structures, allow refined characterization of the central kinetic and chemical steps of the reaction cycle. Quinonoid intermediate formation occurs with an apparent pKa of 7.7 ± 0.1, which is assigned to His-207 acid-catalyzed decarboxylation of the α-amino-β-ketoadipate intermediate to form an enol that is in rapid equilibrium with the 5-aminolevulinate-bound quinonoid species. Quinonoid intermediate decay occurs in two kinetic steps, the first of which is acid-catalyzed with a pKa of 8.1 ± 0.1, and is assigned to protonation of the enol by Lys-313 to generate the product-bound external aldimine. The second step of quinonoid decay defines kcat and is relatively pH-independent and is assigned to opening of the active site loop to allow ALA dissociation. The data support important refinements to both the chemical and kinetic mechanisms and indicate that 5-aminolevulinate synthase operates under the stereoelectronic control predicted by Dunathan's hypothesis. 5-Aminolevulinate synthase (ALAS) 3The abbreviations used are: ALAS, 5-aminolevulinate synthase; ALA, 5-aminolevulinate; AMPSO, 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid; KIE, kinetic isotope effect; mALAS-2, mature form of the murine erythroid specific isoform of aminolevulinate synthase; MOPS, 3-[N-morpholino]propanesulfonic acid; PLP, pyridoxal 5′-phosphate; TAPS, N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid. is a homodimeric pyridoxal 5′-phosphate (PLP)-dependent enzyme that is evolutionarily related to transaminases and catalyzes the first committed step of tetrapyrrole synthesis in non-plant eukaryotes, as well as the α-subclass of purple bacteria (1Christen P. Mehta P.K. Chem. Rec. 2001; 1: 436-447Crossref PubMed Scopus (162) Google Scholar, 2Ferreira G.C. Zhang J.S. Cell Mol. Biol. (Noisy-le-grand). 2002; 48: 827-833PubMed Google Scholar, 3Schulze J.O. Schubert W.D. Moser J. Jahn D. Heinz D.W. J. Mol. Biol. 2006; 358: 1212-1220Crossref PubMed Scopus (42) Google Scholar). Many organisms, including animals and some bacteria, are known to encode two genetically distinct ALAS genes. In animals one of these genes is expressed exclusively in developing erythrocytes, and mutations in the human erythroid-specific ALAS are correlated with hereditary X-linked sideroblastic anemia, a blood disorder characterized by iron-overloaded, heme-deficient red cells (4Bottomley S.S. Greer J.F. Lukens J. Rodgers J.N. Paraskevas G.M. Glader R.B. Wintrobe's Clinical Hematology. 2004: 1011-1033Google Scholar). PLP-dependent enzymes catalyze a wide variety of reactions, including transaminations, decarboxylations, racemizations, and retro-aldol cleavages (5Eliot A.C. Kirsch J.F. Annu. Rev. Biochem. 2004; 73: 383-415Crossref PubMed Scopus (666) Google Scholar, 6Toney M.D. Arch. Biochem. Biophys. 2005; 433: 279-287Crossref PubMed Scopus (226) Google Scholar). In the vast majority of cases the biochemical versatility of PLP can be rationalized in terms of a single property of the cofactor, the potential to act as an electron sink, and stabilize negative charge at the α-carbon of the substrate amino acid. Electrons from cleaved bonds of the covalently bound substrate can delocalize into the conjugated pyridine ring system to form quinonoid intermediates, which are often sufficiently stable to be spectroscopically observable and are characterized by strong absorption maxima of ∼500 nm. These and other changes in the spectroscopic properties of the PLP cofactor during partial or complete reaction cycles can provide important insights into the chemical and kinetic properties of these enzymes. The generally accepted chemical mechanism of ALAS is outlined in Scheme 1 (7Kikuchi G. Kumar A. Talmage P. Shemin D. J. Biol. Chem. 1958; 233: 1214-1219Abstract Full Text PDF PubMed Google Scholar, 8Nandi D.L. J. Biol. Chem. 1978; 253: 8872-8877Abstract Full Text PDF PubMed Google Scholar, 9Zaman Z. Jordan P.M. Akhtar M. Biochem. J. 1973; 135: 257-263Crossref PubMed Scopus (50) Google Scholar). This reaction mechanism appears unnecessarily complex, because decarboxylation of glycine followed by condensation with succinyl-CoA to generate ALA directly would represent a more straightforward synthesis. This possibility is negated, however, by experimental data indicating that the initial and final steps of the chemical mechanism involve proton transfers from and to the amino acid α-carbon, in analogy to aminotransferases (9Zaman Z. Jordan P.M. Akhtar M. Biochem. J. 1973; 135: 257-263Crossref PubMed Scopus (50) Google Scholar, 10Akhtar M. Abboud M.M. Barnard G. Jordan P. Zaman Z. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1976; 273: 117-136Crossref PubMed Scopus (29) Google Scholar, 11Hunter G.A. Ferreira G.C. Biochemistry. 1999; 38: 3711-3718Crossref PubMed Scopus (35) Google Scholar). The existence of the α-amino-β-ketoadipate intermediate (IV) has not been definitively confirmed, but the occurrence of the corresponding intermediate in both amino-oxononanoate synthase (12Kerbarh O. Campopiano D.J. Baxter R.L. Chem. Commun. (Camb.). 2006; : 60-62Crossref PubMed Google Scholar) and ketobutyrate ligase (13Schmidt A. Sivaraman J. Li Y. Larocque R. Barbosa J.A. Smith C. Matte A. Schrag J.D. Cygler M. Biochemistry. 2001; 40: 5151-5160Crossref PubMed Scopus (76) Google Scholar), enzymes that are structurally and mechanistically homologous with ALAS, suggest it is on the ALAS reaction pathway. Notwithstanding this supporting evidence, the mechanistically unusual sequential cleavage of not one but two bonds to the amino acid substrate α-carbon is another factor casting doubt on the accuracy and sufficiency of Scheme 1. Cleavage of two α-carbon bonds is particularly difficult to reconcile with the stereoelectronic control hypothesis explaining the remarkable reaction-type specificity of PLP-dependent enzymes (6Toney M.D. Arch. Biochem. Biophys. 2005; 433: 279-287Crossref PubMed Scopus (226) Google Scholar, 14Dunathan H.C. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 712-716Crossref PubMed Scopus (343) Google Scholar). Based on earlier work by Corey (15Corey R.A.E.J.S. J. Am. Chem. Soc. 1956; 78: 6269-6278Crossref Scopus (209) Google Scholar), Dunathan proposed that in order for efficient electron delocalization into the conjugated ring system to occur the bond to be cleaved must be oriented perpendicular to the plane of the PLP ring (14Dunathan H.C. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 712-716Crossref PubMed Scopus (343) Google Scholar). This orthogonal orientation most closely aligns the sigma orbitals of the bond to be cleaved with the pi orbitals of the conjugated ring system, and because these orbitals must coalesce and rehybridize in the transition state, the said orientation would theoretically provide a pathway with lowered activation energy by bringing the reactants part way along the reaction coordinate toward the transition state. The contour and magnitude of the free energy changes associated with adoption of this stereoelectronic orientation are as yet unknown, but experimental (16Fogle E.J. Liu W. Woon S.T. Keller J.W. Toney M.D. Biochemistry. 2005; 44: 16392-16404Crossref PubMed Scopus (33) Google Scholar) and computational (17Tsai M.D. Weintraub H.J. Byrn S.R. Chang C. Floss H.G. Biochemistry. 1978; 17: 3183-3188Crossref PubMed Scopus (25) Google Scholar) studies have yielded estimates of 3–8 kcal/mol, corresponding to rate accelerations of up to a millionfold. In order for sequential cleavage of two of the glycine α-carbon bonds to occur in strict adherence with Dunathan's hypothesis, the stereochemistry involved in the ALAS mechanism described by Scheme 1 would imply torsion about the aldimine linkage of ∼60° at some point during the time interval between formation of the two quinonoid intermediates. Alternatively, decarboxylation of the reaction intermediate (IV) might initially occur through the β-keto group rather than into the cofactor ring, as has been suggested for amino-oxononanoate synthase (18Alexeev D. Baxter R.L. Campopiano D.J. Kerbarh O. Sawyer L. Tomczyk N. Watt R. Webster S.P. Org. Biomol. Chem. 2006; 4: 1209-1212Crossref PubMed Scopus (33) Google Scholar), or ALAS may represent a notable exception to Dunathan's hypothesis. Resolution of these ambiguities would provide insights not only into the ALAS catalytic mechanism, but also the scope and stringency of Dunathan's stereoelectronic control hypothesis. A model for catalysis by ALAS involving interconversion between "open" and "closed" conformations has been proposed based on kinetic data (19Hunter G.A. Ferreira G.C. J. Biol. Chem. 1999; 274: 12222-12228Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Specifically, it has been suggested that a portion of the intrinsic binding energy for the second substrate, succinyl-CoA, is sacrificed to shift the equilibrium toward the closed conformation where catalysis occurs, and turnover is limited by reversion to the open conformation, which is associated with release of ALA from the enzyme. The recently solved crystal structures of the Rhodobacter capsulatus ALAS holoenzyme, along with structures in which glycine or succinyl-CoA are bound at the active site, appear to be remarkably consistent with this kinetically derived model (20Astner I. Schulze J.O. van den Heuvel J. Jahn D. Schubert W.D. Heinz D.W. EMBO J. 2005; 24: 3166-3177Crossref PubMed Scopus (164) Google Scholar). The active site is located at the bottom of a channel at the interface between the subunits, and a short stretch of highly conserved amino acids near the C terminus closes over the active site in the substrate-bound structures but adopts a more open conformation in one subunit of the holoenzyme structure. The structures suggest that this "loop" closes over the active site and locks succinyl-CoA in the proper juxtaposition for catalysis and, subsequently, opens to allow ALA dissociation. The succinyl-CoA-bound structure indicates that molecular recognition of this substrate includes interactions that bridge the apposing enzyme domains and apparently stabilize the closed conformation. The ALAS active site contains distinct features that should help further clarify the catalytic mechanism. The PLP cofactor is oriented such that the plane of the pyridinium ring is nearly perpendicular to the active site channel, and the pro-R proton of bound glycine faces down toward the protein core perpendicular to the plane of the cofactor ring and toward the side chain of the catalytic lysine residue, whereas the carboxyl group is oriented ∼30° out of the plane of the cofactor ring. The side of the cofactor ring facing the active site channel is not entirely solvent-exposed, because it is overlaid by a histidine residue that forms part of a conserved network of hydrogen bonds that bridge the pyridinium ring nitrogen of the cofactor through four amino acid side chains to the thioester carbonyl of succinyl-CoA (Fig. 1). This carbonyl is conserved in the product ALA and appears to be required to form a quinonoid intermediate, based on the absence of a quinonoid upon binding of aminopentanoate, an ALA analog in which the carbonyl carbon is saturated (21Zhang J. Ferreira G.C. J. Biol. Chem. 2002; 277: 44660-44669Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The observation that these two potential electron sinks are bridged by a conserved hydrogen bonding network suggests the possibility of a functional connectivity. The kinetic mechanism involves ordered binding of glycine and succinyl-CoA, followed by formation of a quinonoid intermediate, and product release, which is rate-determining (8Nandi D.L. J. Biol. Chem. 1978; 253: 8872-8877Abstract Full Text PDF PubMed Google Scholar, 19Hunter G.A. Ferreira G.C. J. Biol. Chem. 1999; 274: 12222-12228Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 21Zhang J. Ferreira G.C. J. Biol. Chem. 2002; 277: 44660-44669Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). It has been proposed that single turnover stopped-flow experiments might allow discrimination of the two putative quinonoid intermediates and provide a basis for detailed characterization of the intermediate steps of the catalytic cycle (19Hunter G.A. Ferreira G.C. J. Biol. Chem. 1999; 274: 12222-12228Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 21Zhang J. Ferreira G.C. J. Biol. Chem. 2002; 277: 44660-44669Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). To elucidate the chemistry and kinetics of the transient reaction steps involved in conversion of substrates into products, and reconcile the available structural and kinetic data with Dunathan's hypothesis, we have conducted single and multiple turnover stopped-flow experiments under conditions of varying pH or temperature. The results have been interpreted within the framework of the recently solved crystal structures and support important refinements to the chemical and kinetic mechanisms. Reagents—The following reagents were from Sigma: DEAE-Sephacel, β-mercaptoethanol, PLP, bovine serum albumin, α-ketoglutarate dehydrogenase, α-ketoglutarate, NAD+, thiamin pyrophosphate, succinyl-CoA, ALA-hydrochloride, HEPES free acid, and the bicinchoninic acid protein determination kit. Glycerol, mono- and dibasic potassium phosphate, disodium EDTA dihydrate, ammonium sulfate, magnesium chloride hexahydrate, glycine, [2-2H2]glycine, and sodium hydroxide were purchased from Fisher. Ultrogel AcA-44 was from IBF Biotechnics. SDS-PAGE reagents were purchased from Bio-Rad. Overexpression, Purification, Storage, Handling, and Analysis of mALAS-2—The overexpression, purification, storage, handling, and analysis of mALAS-2 were conducted as described previously (19Hunter G.A. Ferreira G.C. J. Biol. Chem. 1999; 274: 12222-12228Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Protein concentrations were determined by the bicinchoninic acid method using bovine serum albumin as the standard, from which the extinction coefficient for the purified enzyme was estimated as 46,000 liter mol–1 cm–1 at 278 nm. Reported enzyme concentrations are based on a monomeric molecular mass of 56,523 kDa, as calculated from the primary amino acid sequence (22Ferreira G.C. Dailey H.A. J. Biol. Chem. 1993; 268: 584-590Abstract Full Text PDF PubMed Google Scholar). Stopped-flow Spectroscopy—Rapid scanning stopped-flow kinetic measurements were conducted using an OLIS model RSM-1000 stopped-flow spectrophotometer. The dead time of this instrument is ∼2 ms, and the observation chamber optical path length is 4.0 mm. Scans covering the wavelength region 320–550 nm were acquired at a rate of 1000 scans per second. For reactions longer than 3–4 s, the collected scans were averaged to yield either 62.5 or 31.25 scans per second to condense the resulting data files to a size suitable for global fit analyses. In all cases the resulting data files contained 3000–4000 total spectral scans. An external water bath was utilized to control the temperature of the syringes and observation chamber, and the temperature at the syringes and observation chamber was determined with a Fisherbrand –20 to 110 °C thermometer. The concentrations of reactants loaded into the syringes were always 2-fold greater than that reported, such that the reported concentrations represent the final concentrations present in the stopped-flow cell compartment after mixing. Observed rate constants were determined by Robust Global Fitting of the acquired spectral data, using the single value decomposition software provided by OLIS, Inc. (17Tsai M.D. Weintraub H.J. Byrn S.R. Chang C. Floss H.G. Biochemistry. 1978; 17: 3183-3188Crossref PubMed Scopus (25) Google Scholar, 18Alexeev D. Baxter R.L. Campopiano D.J. Kerbarh O. Sawyer L. Tomczyk N. Watt R. Webster S.P. Org. Biomol. Chem. 2006; 4: 1209-1212Crossref PubMed Scopus (33) Google Scholar). Quality of fits was judged by analysis of the calculated residuals, and the simplest mechanism adequate to accurately describe the experimental data was used. Single turnover data were modeled using a three-kinetic-step mechanism as described by Reaction 1, whereas multiple turnover data were modeled using a two-step kinetic mechanism as described by Reaction 2. A→kobs1B→kobs2C→kobs3D1 A→kobs1B→kobs2C2 For each set of experimental conditions the observed rate constants were determined from three or more replicate experiments, and the reported values represent the average and standard error of measurement for each experimental condition. Estimation of both forward and reverse rate constants was accomplished by modeling single wavelength kinetic traces at 510 nm with KinTekSim kinetic simulation software (23Barshop B.A. Wrenn R.F. Frieden C. Anal. Biochem. 1983; 130: 134-145Crossref PubMed Scopus (669) Google Scholar). The eight interior rate constants were allowed to float, while the KD values were held constant as determined separately (19Hunter G.A. Ferreira G.C. J. Biol. Chem. 1999; 274: 12222-12228Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Single Turnover pH Variation Experiments—Single turnover stopped-flow pH studies were conducted at 30 °C under a constant set of background conditions with the buffer and pH as the only variables. One syringe was filled with a solution containing 100 μm purified mALAS-2, 260 mm glycine, 10% (v/v) glycerol, and 100 mm buffer, whereas the other syringe contained 20 μm succinyl-CoA, 10% (v/v) glycerol, and 100 mm buffer. The buffers utilized were: MES, pH 6.0–6.5; MOPS, pH 6.6–7.0; HEPES, pH 7.1–7.8; TAPS, pH 7.9–8.5; and AMPSO, pH 8.6–9.2. These conditions yielded a solution of 100 μm mALAS-2-glycine complex reacting with an equivalent volume of a solution containing 20 μm succinyl-CoA such that the final concentrations in the observation chamber were 50 μm mALAS-2-glycine complex, 10 μm succinyl-CoA, 10% (v/v) glycerol, and 100 mm buffer. The presence of glycerol was essential to maintain enzyme solubility at lower pH values. Multiple Turnover pH Variation Experiments—Multiple turnover stopped-flow pH studies were conducted at 20 °C. Again, the only variables were the buffer and pH. One of the syringes was loaded with a solution containing 80 μm mALAS-2, 260 mm glycine, 10% (v/v) glycerol, and 100 mm buffer, whereas the other syringe contained 200 μm succinyl-CoA, 10% (v/v) glycerol, and 100 mm buffer. Upon mixing equivalent volumes from each of the two syringes the final concentrations of enzyme and substrates in the observation chamber were halved such that the reaction observed was between 40 μm mALAS-2-glycine complex and 100 μm succinyl-CoA. The buffers and pH ranges were the same as described for the single turnover experiments. For both single and multiple turnover experiments the shots from each set of conditions were collected into a clean waste syringe, and the final pH was verified by direct reading using an Accumet AR15 pH meter. The syringes and observation chamber were flushed with 3–4 syringe volumes of purified water between each set of experimental conditions to remove residual buffer between experiments. pH determinations were conducted at the same temperature as that used in the stopped-flow experiments. Apparent pKa values for quinonoid intermediate formation and the first step of quinonoid intermediate decay were determined by fitting the observed rate constants as a function of pH to Equation 1 using SigmaPlot graphing software, kobs=A(1+10pH−pKa)+B3 where kobs is the observed rate constant, A is the theoretical difference in the observed rate constants associated with the protonated and deprotonated species, pKa is the pH at which the ionizing group is 50% protonated, and B is the rate of reaction when the ionizing group is 100% deprotonated. Single Turnover Temperature Variation Studies—Single turnover temperature variation studies were conducted using conditions identical to those described for the single turnover pH studies, with the pH held constant at 8.1 and the temperature varied from 5 to 35 °C in 5 °C increments. A pH of 8.1 was considered to represent a reasonable compromise between the low signal amplitudes observed at lower pH values and the extended reaction times required at higher pH values, as seen in Fig. 3. The formation of condensate on the stopped-flow cell at low temperatures was prevented by purging the observation chamber with nitrogen during the experiments. Thermodynamic Activation Parameters—Temperature dependence data were fit to the Arrhenius equation (Equation 2), ln(kobs)=−EaR1T+ln(A)4 where Ea is the activation energy, R is the universal gas constant, T is the absolute temperature, and A is the frequency factor. Activation energies were calculated by multiplying the slopes of the Arrhenius plots by the negative reciprocal of the gas constant. Enthalpies of activation were then obtained using the activation energy and Equation 3, where ΔH‡ is the enthalpy of activation. ΔH‡=Ea−RT5 Observed free energies of activation were determined separately at 30 °C and pH 7.5 using Equation 4, which relates the free energy of activation to the observed rate constant. ΔG‡=−RTln(kobsh/kBT)6 Where ΔG‡ is the observed free energy of activation, kB is Boltzmann's constant, and h is Planck's constant. Observed entropies of activation were estimated using Equation 5 and the derived free energies and enthalpies of activation as described above. ΔS‡=−ΔG‡−ΔH‡T7 Kinetic Isotope Effects with Dideuteroglycine—Single turnover studies to determine the kinetic isotope effects resulting from substitution of glycine with dideuteroglycine were conducted at pH 7.5 and 30 °C. One syringe contained 200 μm enzyme and 320 mm glycine (or dideuteroglycine) in 50 mm HEPES, pH 7.5, and 10% glycerol, whereas the other syringe contained 40 μm succinyl-CoA in the same buffer. Glycine or dideuteroglycine was added last, and the experiments were conducted immediately thereafter. Structural Analyses—The protein data base files 2BWN, 2BWO, and 2BWP, corresponding to the holoenzyme, succinyl-CoA bound, and glycine-bound R. capsulatus ALAS crystal structures, were used as models for the mALAS-2 catalytic core (20Astner I. Schulze J.O. van den Heuvel J. Jahn D. Schubert W.D. Heinz D.W. EMBO J. 2005; 24: 3166-3177Crossref PubMed Scopus (164) Google Scholar). Superpositioning of crystal structures and hydrogen bond determinations were accomplished using Deepview/Swiss-PdbViewer software (24Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4505) Google Scholar, 25Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9586) Google Scholar). Modeling of the α-amino-β-ketoadipate intermediate into the active site was accomplished using PyMOL software 4W. L. DeLano (2002) PyMOL, DeLano Scientific, San Carlos, CA. and drawing the α-amino-β-ketoadipate intermediate such that the hydrogen bonds to the succinylmoiety of succinyl-CoA were preserved in the corresponding atoms of the intermediate. The major spectral change observed during single turnover stopped-flow absorbance studies was the formation and decay of a quinonoid intermediate with an absorbance maximum of ∼510 nm. During early pilot experiments a variety of kinetic mechanisms was examined as prospective models for fitting the global data, with the goal of ascertaining the simplest kinetic mechanism that accurately described the experimental data. The single turnover kinetic profiles were found to be best described by the three-step sequential mechanism represented by Reaction 1. The three reaction steps were visually observable in single wavelength traces of quinonoid intermediate formation and decay, as illustrated in Fig. 2, which is from a representative single turnover experiment at pH 7.5 and 30 °C. In Fig. 2A, the time course at 510 nm was overlaid with the global fit of the spectral data at the same wavelength using Reaction 1. The reaction was observed to involve a single kinetic step of quinonoid intermediate formation followed by two decay steps. Other early experiments indicated the rate and amplitude of the second step of quinonoid intermediate decay was dependent upon glycine concentration, a result that was interpreted as a reflection of competitive binding between the ALA produced during the single turnover, which binds relatively tightly and forms a quinonoid intermediate, and glycine, which does not bind tightly and does not form a quinonoid intermediate. To ensure that the second step of quinonoid intermediate decay accurately approximated the rate of ALA release from the enzyme, excess glycine was essential to trap the enzyme in the glycine-bound form at the reaction end point. In the experiments reported here the concentration of glycine was maintained at seven or more times the spectrophotometrically determined dissociation constant, whereas the concentration of ALA formed was never more than approximately one-half the dissociation constant. The calculated spectra for each of the four kinetic species from the global fitting are graphed in Fig. 2B. The starting and ending spectra were similar, whereas the two intermediary spectra, corresponding to the two kinetic intermediates, differed primarily in the magnitude of quinonoid absorbance. The global fit indicated that the quinonoid intermediate arose from the 420 nm peak with an isosbestic point at ∼442 nm. A video of the time course entitled "mALAS-2 single turnover" is provided as supplemental data. The first 5 s of the modeled time courses for each of the four species indicate that the two intermediates were fully formed within 250 and 1600 ms, respectively. The observed rate constants for the three steps of the reaction were 5.3 ± 0.6/s, 2.8 ± 0.3/s, and 0.074 ± 0.005/s. The pH dependence of the single turnover reaction kinetics at 30 °C was investigated. These studies indicated that pH changes had substantial effects on the observed rate constants for quinonoid intermediate formation and the first step of quinonoid decay, whereas the second step of quinonoid decay was relatively pH-independent over the range tested (Fig. 3). In Fig. 3A representative 510 nm single-wavelength kinetic traces were extracted and overlaid with the best global fit to the spectral data at this wavelength. The observed rate constants for all three kinetic steps increased as the pH was lowered, although only the first step of quinonoid intermediate decay could be accurately described by Equation 1 for an acid-catalyzed reaction involving transfer of a single proton. This set of experiments suggested quinonoid intermediate formation was also acid-catalyzed, but the small proportion of data points associated with this kinetic phase, particularly at lower pH values where the signal amplitude was also low, led to large standard errors. Additionally, it was not possible to estimate an acid-catalyzed end point for quinonoid intermediate formation, due to increasing instability of the enzyme as the pH was dropped below 7.0. The single turnover pH dependence of the three phases are plotted in Fig. 3B. A fit of the first step of quinonoid decay to Equation 1 resulted in an apparent kinetic pKa of 8.1 ± 0.1. This value was virtually identical to the previously determined apparent spectroscopic pKa for Lys-313-catalyzed abstraction of the C-4 pro-R proton of bound ALA to form a quinonoid intermediate (11Hunter G.A. Ferreira G.C. Biochemistry. 1999; 38: 3711-3718Crossref PubMed Scopus (35) Google Scholar). The difficulties associated with accurate determination of the pH dependence of quinonoid formation were overcome by switching to multiple turnover conditions with excess succinyl-CoA at 20 °C. Under multiple turnover conditions quinonoid intermediate formation is followed by a single step representing decay into the steady state (19Hunter G.A. Ferreira
Referência(s)