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• W2124012017 abstract "In addition to α, β-elimination of l-cysteine, Treponema denticola cystalysin catalyzes the racemization of both enantiomers of alanine accompanied by an overall transamination. Lys-238 and Tyr-123 or a water molecule located on the si and re face of the cofactor, respectively, have been proposed to act as the acid/base catalysts in the proton abstraction/donation at Cα/C4′ of the external aldimine. In this investigation, two site-directed mutants, K238A and Y123F, have been characterized. The Lys → Ala mutation results in the complete loss of either lyase activity or racemase activity in both directions or transaminase activity toward l-alanine. However, the K238A mutant is able to catalyze the overall transamination of d-alanine, and only d-alanine is the product of the reverse transamination. For Y123F the kcat/Km is reduced 3.5-fold for α, β-elimination, whereas it is reduced 300–400-fold for racemization. Y123F has ∼18% of wild type transaminase activity with l-alanine and an extremely low transaminase activity with d-alanine. Moreover, the catalytic properties of the Y124F and Y123F/Y124F mutants rule out the possibility that the residual racemase and transaminase activities displayed by Y123F are due to Tyr-124. All these data, together with computational results, indicate a two-base racemization mechanism for cystalysin in which Lys-238 has been unequivocally identified as the catalyst acting on the si face of the cofactor. Moreover, this study highlights the importance of the interaction of Tyr-123 with water molecules for efficient proton abstraction/donation function on the re face. In addition to α, β-elimination of l-cysteine, Treponema denticola cystalysin catalyzes the racemization of both enantiomers of alanine accompanied by an overall transamination. Lys-238 and Tyr-123 or a water molecule located on the si and re face of the cofactor, respectively, have been proposed to act as the acid/base catalysts in the proton abstraction/donation at Cα/C4′ of the external aldimine. In this investigation, two site-directed mutants, K238A and Y123F, have been characterized. The Lys → Ala mutation results in the complete loss of either lyase activity or racemase activity in both directions or transaminase activity toward l-alanine. However, the K238A mutant is able to catalyze the overall transamination of d-alanine, and only d-alanine is the product of the reverse transamination. For Y123F the kcat/Km is reduced 3.5-fold for α, β-elimination, whereas it is reduced 300–400-fold for racemization. Y123F has ∼18% of wild type transaminase activity with l-alanine and an extremely low transaminase activity with d-alanine. Moreover, the catalytic properties of the Y124F and Y123F/Y124F mutants rule out the possibility that the residual racemase and transaminase activities displayed by Y123F are due to Tyr-124. All these data, together with computational results, indicate a two-base racemization mechanism for cystalysin in which Lys-238 has been unequivocally identified as the catalyst acting on the si face of the cofactor. Moreover, this study highlights the importance of the interaction of Tyr-123 with water molecules for efficient proton abstraction/donation function on the re face. Treponema denticola cystalysin catalyzes the pyridoxal 5′-phosphate (PLP) 1The abbreviations used are: PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate.1The abbreviations used are: PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate.-dependent α,β-elimination of l-cysteine to generate pyruvate, ammonia, and H2S (1Chu L. Ebersole J.L. Kurzban G.P. Holt S.C. Infect. Immun. 1997; 65: 3231-3238Crossref PubMed Google Scholar). It has been shown that several sulfur- and non-sulfur-containing amino acids as well as disulfidic amino acids also serve as substrates for this reaction (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (41) Google Scholar). The protein is composed of two identical subunits, each of which comprises 399 amino acid residues. The three-dimensional structure of the enzyme has been solved to 1.9 Å (3Krupka H.L. Huber R. Holt S.C. Clausen T. EMBO J. 2000; 19: 3168-3178Crossref PubMed Scopus (66) Google Scholar). The PLP cofactor in the active site forms a Schiff base with the ϵ-amino group of Lys-238 of cystalysin (3Krupka H.L. Huber R. Holt S.C. Clausen T. EMBO J. 2000; 19: 3168-3178Crossref PubMed Scopus (66) Google Scholar). Through site-directed mutagenesis, spectroscopic, and kinetic studies, we have shown recently that Lys-238 is an essential residue for α,β-elimination catalyzed by the enzyme cystalysin. In addition to strengthening the coenzyme binding and facilitating transimination, this residue seems to participate as a general base abstracting the Cα proton from the substrate and possibly as a general acid, protonating the β-leaving group (4Bertoldi M. Cellini B. D'Aguanno S. Borri Voltattorni C. J. Biol. Chem. 2003; 278: 37336-37343Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar).In a recent paper we have shown that, in addition to the lyase activity, the enzyme exhibits an alanine racemase activity accompanied by an overall transaminase activity toward both enantiomers of alanine (5Bertoldi M. Cellini B. Paiardini A. Di Salvo M. Borri Voltattorni C. Biochem. J. 2003; 371: 473-483Crossref PubMed Scopus (18) Google Scholar). Considering that racemization is a side reaction of cystalysin, it occurs with a significant kcat (∼1 s–1), even if it is ∼1000-fold lower than that of alanine racemase (6Sun S. Toney M.D. Biochemistry. 1999; 38: 4058-4065Crossref PubMed Scopus (123) Google Scholar). The structure of cystalysin with aminoethoxyvinylglycine has revealed that Lys-238 and Tyr-123, located on the si and re face of the cofactor, respectively, are structurally well positioned to be general acid/base catalysts in a two-base racemization mechanism (3Krupka H.L. Huber R. Holt S.C. Clausen T. EMBO J. 2000; 19: 3168-3178Crossref PubMed Scopus (66) Google Scholar). A similar active site architecture has been reported for the crystal structure of alanine racemase from Bacillus stearothermophilus with an alanine phosphonate in which PLP is bound such that Tyr-265′ faces the si side of the cofactor and Lys-39 the re side (7Stamper G.F. Morollo A.A. Ringe D. Biochemistry. 1998; 37: 10438-10445Crossref PubMed Scopus (97) Google Scholar). For alanine racemase a two-base racemization mechanism involving Tyr-265′ and Lys-39 has been demonstrated by site-directed mutagenesis studies (8Watanabe A. Yoshimura T. Mikami B. Esaki N. J. Biochem. 1999; 126: 781-786Crossref PubMed Scopus (69) Google Scholar, 9Watanabe A. Kurokava Y. Yoshimura T. Kurihara T. Soda K. Esaki N. J. Biol. Chem. 1999; 274: 4189-4194Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). On the basis of structural and kinetic data of cystalysin, we have proposed that racemization occurs by a two-base mechanism. Lys-238 and Tyr-123 have been suggested as possible candidates for general acid-base catalysis, even though the possibility has not been excluded that the crystallographic water molecule located on the re side could serve as the acid/base catalyst (5Bertoldi M. Cellini B. Paiardini A. Di Salvo M. Borri Voltattorni C. Biochem. J. 2003; 371: 473-483Crossref PubMed Scopus (18) Google Scholar). According to the generally accepted mechanism as well as on the basis of our studies, alanine racemase reaction is proposed to proceed in the following manner as shown in Scheme 1: (i) transaldimination between Lys-238 bound with PLP (I) and the α-amino group of alanine to produce an external aldimine II; (ii) abstraction of the α-hydrogen from alanine to produce a resonance-stabilized quinonoid intermediate III; (iii) reprotonation at the α-carbon of the quinonoid intermediate III on the side opposite to that where the α-hydrogen was abstracted; and (iv) the second transaldimination between IV and Lys-238 to release the product enantiomer of alanine. An equivalent route can be delineated for d-alanine. The transamination catalyzed by cystalysin is probably attained through the sequences I → II → III → VI or V → IV → III → VI for l-and d-alanine, respectively.As a first step in testing the hypothetical function of Lys-238 and Tyr-123 in the catalysis of cystalysin, the enzymatic activities of the K238A and Y123F mutants were determined. The results provide evidence in support of our previously proposal (5Bertoldi M. Cellini B. Paiardini A. Di Salvo M. Borri Voltattorni C. Biochem. J. 2003; 371: 473-483Crossref PubMed Scopus (18) Google Scholar) of a two-base racemization mechanism of both of the enantiomers of alanine. However, only the PLP binding lysine, Lys-238, has been unequivocally identified as the catalyst acting on the si face of the cofactor. In fact, the Y123F mutant exhibits poor racemase and transaminase activities. A previous modeling study of the binding modes of the enantiomers of alanine to wild-type cystalysin has indicated that Tyr-124 is too far from the α-hydrogen of the substrate (4.1 Å) for the α-proton abstraction (5Bertoldi M. Cellini B. Paiardini A. Di Salvo M. Borri Voltattorni C. Biochem. J. 2003; 371: 473-483Crossref PubMed Scopus (18) Google Scholar). However, it cannot be excluded that the geometry of the active site could be altered upon replacement of Tyr-123 by phenylalanine. Thus, the possibility that the catalytic function of Tyr-123 could be replaced by Tyr-124 has been addressed by the spectroscopic and kinetic characterization of the Y124F and Y123F/Y124F mutants. These studies show that Tyr-124 cannot replace Tyr-123. Therefore, the role of Tyr-123 and/or water molecules in performing the proton abstraction/donation function on the re face of the cofactor is discussed.EXPERIMENTAL PROCEDURESMaterials—PLP, pyridoxamine 5′-phosphate (PMP), β-chloro-l-alanine, l- and d-alanine, NAD+, NADH, pyruvate, rabbit muscle l-lactic dehydrogenase, alanine dehydrogenase in 50% glycerol, d-amino acid oxidase, and isopropyl β-d-thiogalactopyranoside were from Sigma. All other chemicals were of the highest grade commercially available.Site-directed Mutagenesis—All mutant forms of cystalysin were made on the wild-type construct pUC18:hly (10Chu L. Burgum A. Kolodrubetz D. Holt S.C. Infect. Immun. 1995; 63: 4448-4455Crossref PubMed Google Scholar) using the Quik-Change™ site-directed mutagenesis kit from Stratagene (La Jolla, CA). The kit employs double-stranded DNA as the template, two complementary oligonucleotide primers containing the desired mutation, and DpnI endonuclease to digest the parental DNA template. Oligonucleotides were synthesized by MWG-Biotech AG (Anzinger, Germany). The Y123F and Y124F mutants and the double mutant (Y123F/Y124F) of cystalysin were produced using as primers 5′-CATTATCATCACACCGGTTTTTTATCCTTTCTTTAT-3′, 5′-CATTATCATCACACCGGTTTATTTTCCTTTCTTTATGGC-3′, 5′-CATTATCATCACACCGGTTTTTTTTCCTTTCTTTAT-3′, and their complementary oligonucleotides, respectively. The mutated bases are underlined.The coding regions of the mutated hly gene were sequenced to confirm the mutations. Escherichia coli strain DH5α cells were transformed and used for expression.Expression and Purification of Mutant Cystalysin—The conditions used for expression of the mutant proteins in E. coli were as described for the wild-type enzyme (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (41) Google Scholar). The wild-type and mutant forms of cystalysin were purified to homogeneity with the procedure described previously (4Bertoldi M. Cellini B. D'Aguanno S. Borri Voltattorni C. J. Biol. Chem. 2003; 278: 37336-37343Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The K238A mutant was obtained as reported previously (4Bertoldi M. Cellini B. D'Aguanno S. Borri Voltattorni C. J. Biol. Chem. 2003; 278: 37336-37343Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The protein concentration in all cystalysin samples was determined by absorbance spectroscopy using a previously determined extinction coefficient of 1.27 × 105m–1 cm–1 at 280 nm (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (41) Google Scholar). The PLP content of wild-type and mutant enzymes was determined by releasing the coenzyme in 0.1 m NaOH and by using ϵ = 6600 m–1 cm–1 at 388 nm (11Peterson E.A. Sober H.A. J. Am. Chem. Soc. 1954; 76: 169-175Crossref Scopus (307) Google Scholar).Enzyme Activity Assays—The α,β-eliminase activity of cystalysin was measured by the spectrophotometric assay coupled with lactate dehydrogenase as described previously (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (41) Google Scholar). The conversion of alanine in the l → d or d → l direction was measured using d-amino acid oxidase and lactate dehydrogenase or l-alanine dehydrogenase as the coupling enzymes, respectively, as reported previously (5Bertoldi M. Cellini B. Paiardini A. Di Salvo M. Borri Voltattorni C. Biochem. J. 2003; 371: 473-483Crossref PubMed Scopus (18) Google Scholar). The detection and quantification of PLP and PMP content were performed using the high pressure liquid chromatography procedure described previously (12Bertoldi M. Borri Voltattorni C. Biochem. J. 2000; 352: 533-538Crossref PubMed Scopus (38) Google Scholar). To determine the kinetic parameters of the catalytic activities, the assays were performed as indicated above using a fixed amount of enzyme, whereas the substrate concentration was varied from 0.02 to 10 mm and from 0.15 to 500 mm for eliminase and racemase or transaminase activities, respectively. The experimental data were fit into the Michaelis-Menten equation to determine Km and kcat values.Binding Affinity of the Y123F, Y124F, and Y123F/Y124F Mutants for the PLP Cofactor—The apparent equilibrium constant for the dissociation of PLP from Y123F, Y124F, and Y123F/Y124F was determined by measuring the α,β-eliminase activity of the apoenzymes (0.6 μm) in the presence of PLP ranging from 0.01 to 9 μm. The Kd value of the enzyme-coenzyme complex was obtained using a tight binding hypothesis according to Equation 1, shown below,Y=Ymax[E]t+[PLP]t+Kd−([E]t+[PLP]t+Kd)2−4[E]t[PLP]t2[E]t(Eq. 1) where [E]t and [PLP]t represent the total concentrations of the cystalysin mutant dimer and PLP, respectively, Y refers to the enzymatic activity change at various PLP concentrations, and Ymax refers to the enzymatic activity when all enzyme molecules are complexed with a coenzyme.Molecular Modeling—The BUILDER package from Insight II (version 2000; MSI, Los Angeles, CA) was used to model the d-alanine-PLP and l-alanine-PLP conjugates, using the PLP-serine complex present in the crystal structure of serine hydroxymethyltransferase from B. stearothermophilus (13Trivedi V. Gupta A. Jala V.R. Saravanan P. Rao G.S.J. Rao N.A. Savithri H.S. Subramanja H.S. J. Biol. Chem. 2002; 277: 17161-17169Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) as template. Tyr-123 and Tyr-124 from the crystal structure of cystalysin (Protein Data Bank code 1C7O) in its dimeric form were then mutated to phenylalanine to obtain the single Y123F and the double Y123F/Y124F mutants using the BIOPOLYMER package from Insight II. Both complexes were positioned into the modified active sites following the binding mode of aminoethoxyvinylglycine (3Krupka H.L. Huber R. Holt S.C. Clausen T. EMBO J. 2000; 19: 3168-3178Crossref PubMed Scopus (66) Google Scholar).All minimizations were carried out using the Cff91 force field as implemented in Discover 2.9 and the Analysis package of Insight II. Active site solvent molecules, as present in the crystal structure, were considered. Non-bond terms were truncated at 40 Å (smoothing from 36 Å) with a switching function for van der Waals and electrostatic terms. Because the other water molecules were not explicitly included, a distance-dependent dielectric was used throughout the minimizations. The minimization protocol is described elsewhere (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (41) Google Scholar). Briefly, after an initial minimization performed on the whole system to allow added hydrogens to adjust to the crystallographically defined environment, a gradually decreasing tethering force using the steepest descents and conjugated gradients was applied to the whole system until the system was totally relaxed. The maximum derivative achieved was 0.0001 kcal·mol–1 Å–1. Only the PLP-Ala complex and main chain and side chains of each residue within 20 Å from the external aldimine were free to move.Spectral Measurements—Absorption measurements were made with a Jasco V-550 spectrophotometer. The enzyme solution was drawn through a 0.2-μm filter to reduce light scattering from a small amount of precipitate. Fluorescence spectra were taken with a FP-750 Jasco spectrofluorometer using 5-nm excitation and emission bandwidths at a protein concentration varying from 0.9 to 2.2 μm. Spectra of blanks, i.e. of samples containing all components except cystalysin, were taken immediately before the measurements of samples containing protein. The blank spectra were subtracted from the spectra containing the enzyme. CD spectra were obtained using a Jasco J-710 spectropolarimeter with a thermostatically controlled compartment at 25 °C. For near-UV and visible wavelengths the protein concentration varied from 0.6 to 1 mg/ml in a cuvette with a 1-cm path length. Routinely, four spectra were recorded at a scan speed of 50 nm/min with a bandwidth of 2 nm and were averaged automatically except where indicated. For far-UV measurements the protein concentration was 0.1 mg/ml with a 0.1-cm path length.Curve Fitting analysis—All data analysis for determining the model-derived kinetic parameters was performed by nonlinear curve fitting using MicroCal Origin 3.01 (MicroCal Software, Inc., Northamton, MA).RESULTSWe used site-directed mutagenesis to prepare several variants of cystalysin with single and double replacements at putative active site residues. The purified variants were homogeneous as indicated by a single band on SDS-PAGE with a mobility identical to that of the corresponding wild-type protein. Yields of the mutant enzymes after the standard purification were ∼ 70% with respect to that of the corresponding wild-type enzyme. The effects of amino acid substitution on the catalytic properties were determined to evaluate the function of these residues and gain insight into the mechanism of catalysis. Because the reactions catalyzed by cystalysin proceed through chromophoric intermediates formed between PLP and ligands, we have also examined the effects of amino acid replacement on the spectra of enzyme-ligand intermediates.To test the structural integrity of the cystalysin mutants, we measured their circular dichroism spectra in the far-UV region. No differences have been observed between the spectra of the mutated and the wild-type forms of cystalysin, which indicates that the mutations do not affect the overall secondary structure of cystalysin (data not shown).Spectroscopic Properties of Tyr-123 and Tyr-124 Mutants— As with wild-type, the Y123F, Y124F, and Y123F/Y124F variants bind 2 mol of PLP per dimer. In contrast, apo K238A reconstituted with PLP binds 1 mol of coenzyme per dimer, as reported previously (4Bertoldi M. Cellini B. D'Aguanno S. Borri Voltattorni C. J. Biol. Chem. 2003; 278: 37336-37343Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). As shown in Fig. 1, A and B, the absorbance and CD spectra of Tyr-123 and Tyr-124 mutants in the UV-visible region are largely similar to those of the wild-type enzyme. The wild-type, Y123F, Y124F, and Y123F/Y124F mutant enzymes display optical activity (millidegrees per absorbance unit) values of 70, 79, 70, and 61 millidegrees/A418, respectively. This suggests that the microenvironment of the internal aldimine in the Tyr-123 mutant enzymes is slightly different from that in the wild-type enzyme. For comparison, the absorbance and CD spectra of the apo K238A reconstituted with PLP reported previously (4Bertoldi M. Cellini B. D'Aguanno S. Borri Voltattorni C. J. Biol. Chem. 2003; 278: 37336-37343Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) are also included in Fig. 1, A and B, respectively. The fluorescence of Tyr-123 and Tyr-124 mutants is similar to that of the wild-type. It is of interest that the intensity of the PLP emission fluorescence (excitation at 418 nm) of the Y123F and Y123F/Y124F mutants is 10-fold higher than that of the wild-type cystalysin (data not shown).Fig. 1Absorbance and CD spectra of wild-type and mutants of cystalysin. A, absorption of wild-type (WT)(——), Y123F (····), Y124F (—–), Y123F/Y124F (·–·–·–), and K238A (–––) in 20 mm potassium phosphate buffer, pH 7.4, at a concentration of 4.5 μm. B, CD spectra; the symbols for wild-type, Y123F, Y124F, Y123F/Y124F, and K238A are the same as for panel A.View Large Image Figure ViewerDownload (PPT)Enzyme Activities of Lys-238, Tyr-123, and Tyr-124 Mutants—Steady-state kinetic studies of the primary reaction (α,β-elimination) and of alanine racemase and transaminase activities were performed on the mutant enzymes and compared with those of wild-type. Although as reported previously (4Bertoldi M. Cellini B. D'Aguanno S. Borri Voltattorni C. J. Biol. Chem. 2003; 278: 37336-37343Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) the K238A mutant is inactive in the α,β-elimination of β-chloro-l-alanine, Y123F, Y124F, and Y123F/Y124F mutants display, to varying extents, significant eliminase activity. A summary of the resulting steady-state parameters obtained for the α,β-elimination reaction toward β-chloro-l-alanine is listed in Table I. For both Y123F and Y123F/Y124F mutants, the Km values were ∼10-fold lower with respect to those of the wild-type. Furthermore, the observed kcat values decreased by 25- and 40-fold for Y123F and Y123F/Y124F, respectively, compared with those of the wild-type enzyme, resulting in a ∼ 3.5-fold reduction of the catalytic efficiency (kcat/Km). As seen in Table I, a slight increase was observed in the Km and kcat values for Y124F, leading to a 50% reduction of the catalytic efficiency with respect to wild-type. The results indicate that, unlike the Lys-238 residue, the Tyr-123 and Tyr-124 residues are not critical for the α,β-eliminase activity catalyzed by cystalysin.Table ISteady-state kinetic parameters for α,β -elimination of β -chloro-l-alanine in 20 mm potassium phosphate buffer, pH 7.4, at 25 °CkcatKmkcat/Kms-1mmmm-1 s-1Wild-typeaFrom Ref. 2.59.9 ± 2.31.21 ± 0.1549.5 ± 6.4K238AbFrom Ref. 4.Y123F2.41 ± 0.080.17 ± 0.0314.2 ± 2.5Y124F74 ± 23.2 ± 0.2023.1 ± 1.6Y123F/Y124F1.44 ± 0.040.10 ± 0.0214.4 ± 2.9a From Ref. 2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (41) Google Scholar.b From Ref. 4Bertoldi M. Cellini B. D'Aguanno S. Borri Voltattorni C. J. Biol. Chem. 2003; 278: 37336-37343Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar. Open table in a new tab The kinetic parameters determined for alanine racemase activity reveal that, although alteration of Lys-238 causes a complete loss of racemase activity, the mutation of Tyr-123 results in a 200-fold reduction of kcat. These effects are similar of those observed for the primary reaction with the K238A variant but are significantly more than those observed for the primary reaction with the Y123F variant, where only a 25-fold decrease in kcat is produced. On the contrary, the kinetic parameters for alanine racemase activity are not significantly altered in the Y124F mutant, analogous to what was observed for the α,β-elimination reaction. For the Y123F/Y124F variant the Km values and the kcat for l-and d-alanine were ∼ 10- and 200-fold lower, respectively, with respect to the corresponding values for wild-type (Table II).Table IISteady-state kinetic parameters for racemization of l- and d-alanine in 20 mm potassium phosphate buffer, pH 7.4, at 25 °CkcatKmkcat/Kml-Alad-Alal-Alad-Alal-Alad-Alas-1mmmm-1 s-1Wild-type1.05 ± 0.031.4 ± 0.110 ± 110 ± 10.10 ± 0.010.14 ± 0.02K238AN.D.aN.D., not detectable.N.D.aN.D., not detectable.Y123F0.0058 ± 0.00020.0069 ± 0.000314.3 ± 1.822.4 ± 3.64 × 10-4 ± 5 × 10-53 × 10-4 ± 5 × 10-5Y124F1.96 ± 0.083.16 ± 0.0320 ± 325.3 ± 0.70.1 ± 0.010.125 ± 0.004Y123F/Y124F0.0047 ± 0.00020.0042 ± 0.00011 ± 0.20.76 ± 0.080.0047 ± 0.00090.0056 ± 0.0006a N.D., not detectable. Open table in a new tab Each Lys-238, Tyr-123, and Tyr-124 variant was also screened for the ability to catalyze the transamination of l- and d-alanine. Y124F mutant catalyzes transamination of l- and d-alanine with kcat values of 0.0177 ± 0.0003 and 0.0035 ± 0.0004 min–1, respectively, about half the value corresponding to wild-type (5Bertoldi M. Cellini B. Paiardini A. Di Salvo M. Borri Voltattorni C. Biochem. J. 2003; 371: 473-483Crossref PubMed Scopus (18) Google Scholar). Both Y123F and Y123F/Y124F mutants catalyze the transamination of l-alanine with kcat values 5- and 10-fold lower, respectively, than that of wild-type. On the other hand, under conditions of relatively high enzyme concentration (30 μm), normally saturating substrate concentrations (500 mm), and extensive time courses (2 h), the reaction of these mutants with d-alanine produces 0.51 μm PMP. Considering that under these conditions l-alanine is produced in amounts less than 0.1 Km, it can be assessed that most of the PMP produced is due to the transamination of d-alanine rather than the transamination of l-alanine formed by racemization of d-alanine. It is noteworthy that the K238A mutant does not show detectable transaminase activity toward l-alanine, whereas it is able to catalyze the transamination of d-alanine. Initial velocity data for this reaction were fitted to Michaelis-Menten equation and yielded the kinetic parameters Km = 24.5 ± 3.5 mm and kcat = 0.125 ± 0.004 min–1, resulting in a catalytic efficiency ∼ 8-fold higher than that of wild-type for d-alanine (5Bertoldi M. Cellini B. Paiardini A. Di Salvo M. Borri Voltattorni C. Biochem. J. 2003; 371: 473-483Crossref PubMed Scopus (18) Google Scholar).As with wild-type, K238A and Y123F apomutants are able to convert the PMP form of the enzyme back into the PLP form in the presence of pyruvate. The kinetic parameters for this reaction are listed in Table III. The kcat/Km value of Y123F is reduced by only 2.5-fold as compared with that of wild-type enzyme. This reduction is due to approximately equal magnitude changes in the kcat and Km values. On the contrary, the kcat/Km of the K238A variant is increased by 25-fold as compared with that of the wild-type. The increase in the catalytic efficiency of this mutant is driven by the decrease in Km (30-fold), as the kcat is not appreciably affected. It is also of interest to note that only alanine in the d-form is generated by the K238A variant, whereas l-alanine is formed in amounts ∼2-fold higher than those of d-alanine by the Y123F mutant. The amounts of l- and d-alanine produced from Y123F have been evaluated after 25 min of reaction of 100 μm variant enzyme in the presence of 200 μm PMP and 0.3 mm pyruvate. Thus, on the basis of the kcat values of racemization and reverse transamination of the Y123F variant, it can be inferred that the production of d-alanine is mainly due to transamination rather than the conversion of l- into d-alanine. It should be noted that l- and d-alanine are produced in nearly equivalent amounts when apo wild-type reacts with pyruvate in the presence of PMP. However, given the higher racemization rate in this case, it was not possible to determine whether both enantiomers of alanine are formed by racemization rather than by transamination (5Bertoldi M. Cellini B. Paiardini A. Di Salvo M. Borri Voltattorni C. Biochem. J. 2003; 371: 473-483Crossref PubMed Scopus (18) Google Scholar).Table IIISteady-state kinetic parameters for reverse transamination of wild-type, K238A, and Y123F mutants in 20 mm potassium phosphate buffer, pH 7.4, at 25 °CkcatKmkcat/Kmmin-1mmmm-1 min-1Wild-type0.108 ± 0.0050.19 ± 0.030.57 ± 0.09K238A0.086 ± 0.0050.006 ± 0.00114 ± 2Y123F0.083 ± 0.0010.391 ± 0.0320.21 ± 0.02 Open table in a new tab Binding Affinity of Y123F and Y124F Mutants for the PLP Cofactor—When wild-type or Y124F was mixed with PLP, the regain of eliminase activity occurred within the time required for the manual mixing of PLP and apoenzyme. In contrast, under the same experimental conditions the regain of activity upon the addition of a coenzyme to either apo Y123F or Y123F/Y124F as a function of time reached a near saturation value within 1 h. Titration analysis of the apomutants Y123F, Y124F, and Y123F/Y124F with PLP fitted to the appropriate equation yielded Kd values for the PLP-Y123F, PLP-Y124F, and PLP-Y123F/Y124F complexes equal to 295 ± 60, 164 ± 26, and 137 ± 30 nm, respectively (data not shown). The apparent Kd for the dissociation of PLP from the wild-type and the K238A mutant has been found previously to be 6.6 ± 0.1 (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (41) Google Scholar) and 70 ± 16 nm (4Bertoldi M. Cellini B. D'Aguanno S. Borri Voltattorni C. J. Biol. Chem. 2003; 278: 37336-37343Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), respectiv" @default.
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