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Lysine 238 Is an Essential Residue for α,β-Elimination Catalyzed by Treponema denticola Cystalysin

2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês

10.1074/jbc.m305967200

1083-351X

Mariarita Bertoldi, Barbara Cellini, Simona D’Aguanno, Carla Borri Voltattorni,

Metabolism and Genetic Disorders

Treponema denticola cystalysin is a pyridoxal 5′-phosphate (PLP) enzyme that catalyzes the α,β-elimination of l-cysteine to pyruvate, ammonia, and H2S. Similar to other PLP enzymes, an active site Lys residue (Lys-238) forms an internal Schiff base with PLP. The mechanistic role of this residue has been studied by an analysis of the mutant enzymes in which Lys-238 has been replaced by Ala (K238A) and Arg (K238R). Both apomutants reconstituted with PLP bind noncovalently ∼50% of the normal complement of the cofactor and have a lower affinity for the coenzyme than that of wild-type. Kinetic analyses of the reactions of K238A and K238R mutants with glycine compared with that of wild-type demonstrate the decrease of the rate of Schiff base formation by 103- and 7.5 × 104-fold, respectively, and, to a lesser extent, a decrease of the rate of Schiff base hydrolysis. Thus, a role of Lys-238 is to facilitate formation of external aldimine by transimination. Kinetic data reveal that the K238A mutant is inactive in the α,β-elimination of l-cysteine and β-chloro-l-alanine, whereas K238R retains 0.3% of the wild-type activity. These data, together with those derived from a spectral analysis of the reaction of Lys-238 mutants with unproductive substrate analogues, indicate that Lys-238 is an essential catalytic residue, possibly participating as a general base abstracting the Cα-proton from the substrate and possibly as a general acid protonating the β-leaving group. Treponema denticola cystalysin is a pyridoxal 5′-phosphate (PLP) enzyme that catalyzes the α,β-elimination of l-cysteine to pyruvate, ammonia, and H2S. Similar to other PLP enzymes, an active site Lys residue (Lys-238) forms an internal Schiff base with PLP. The mechanistic role of this residue has been studied by an analysis of the mutant enzymes in which Lys-238 has been replaced by Ala (K238A) and Arg (K238R). Both apomutants reconstituted with PLP bind noncovalently ∼50% of the normal complement of the cofactor and have a lower affinity for the coenzyme than that of wild-type. Kinetic analyses of the reactions of K238A and K238R mutants with glycine compared with that of wild-type demonstrate the decrease of the rate of Schiff base formation by 103- and 7.5 × 104-fold, respectively, and, to a lesser extent, a decrease of the rate of Schiff base hydrolysis. Thus, a role of Lys-238 is to facilitate formation of external aldimine by transimination. Kinetic data reveal that the K238A mutant is inactive in the α,β-elimination of l-cysteine and β-chloro-l-alanine, whereas K238R retains 0.3% of the wild-type activity. These data, together with those derived from a spectral analysis of the reaction of Lys-238 mutants with unproductive substrate analogues, indicate that Lys-238 is an essential catalytic residue, possibly participating as a general base abstracting the Cα-proton from the substrate and possibly as a general acid protonating the β-leaving group. Cystalysin utilizes pyridoxal 5′-phosphate (PLP) 1The abbreviations used are: PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate; PNP, pyridoxine 5′-phosphate; HPLC, high pressure liquid chromatography.1The abbreviations used are: PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate; PNP, pyridoxine 5′-phosphate; HPLC, high pressure liquid chromatography. as its coenzyme and is categorized as a member of the α subfamily of PLP-dependent enzymes that include aspartate aminotransferase. The cDNA from Treponema denticola has been heterologously cloned in Escherichia coli, the recombinant enzyme has been crystallized, and the three-dimensional structure solved (1Krupka H.I. Huber R. Holt S.C. Clausen T. EMBO J. 2000; 19: 3168-3178Crossref PubMed Scopus (64) Google Scholar). The PLP cofactor in the active site forms a Schiff base with the ϵ-amino group of Lys-238 of cystalysin (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). The spectrum of cystalysin exhibits absorption maxima at 418 and 320 nm, in addition to the protein band at 281 nm. The 418-nm band is due to the protonated internal aldimine, whereas the 320-nm band is due to a substituted aldamine. The apparent pK spec of this spectral transition is ∼8.4 (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). Cystalysin catalyzes the α,β-elimination of l-cysteine to generate pyruvate, ammonia, and H2S (3Chu L. Burgum A. Kolodrubetz D. Holt S.C. Infect. Immun. 1995; 63: 4448-4455Crossref PubMed Google Scholar). A catalytic mechanism has been suggested in which, after transaldimination, the released Lys-238 abstracts the Cα proton from the substrate with its deprotonated ϵ-amino group, producing a carbanionic intermediate stabilized as the characteristic quinonoid intermediate. Then, after reaching an optimal position within hydrogen bonding distance to Sγ, Lys-238 protonates the sulfur atom, and the resulting thiol is released. Finally, reverse transaldimination of the PLP aminoacrylate takes place; Lys-238 attacks the C4′ atom, converting the aminoacrylate into an iminoproprionate, which is released and hydrolyzed to pyruvate and ammonia (1Krupka H.I. Huber R. Holt S.C. Clausen T. EMBO J. 2000; 19: 3168-3178Crossref PubMed Scopus (64) Google Scholar). Recently, substrate specificity studies reveal that cystalysin displays catalytic features similar to those of cyste(i)ne desulfhydrase (C-DES). Indeed, several sulfur- and non-sulfur-containing amino acids as well as disulfidic amino acids serve as substrates for cystalysin, with l-djenkolic acid and l-cystine being better substrates than l-cysteine (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). The pH dependence of the kinetic parameters for α,β-elimination indicates that a single ionizing group with a pK value of ∼ 6.6 must be unprotonated to achieve maximum velocity. This pK has been tentatively associated with the ionization of Lys-238 (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). Lysine residues involved in internal aldimines in PLP-dependent enzymes have been proposed to have a multitude of roles. They have been shown to increase the reactivity of the PLP 4′-aldehyde group, facilitating the formation of an external aldimine between the substrate and the PLP cofactor (3Chu L. Burgum A. Kolodrubetz D. Holt S.C. Infect. Immun. 1995; 63: 4448-4455Crossref PubMed Google Scholar, 4Nishimura K. Tanizawa K. Yoshimura T. Esaki N. Futaki S. Manning J.M. Soda K. Biochemistry. 1991; 30: 4072-4077Crossref PubMed Scopus (35) Google Scholar, 5Lu Z. Nagata S. McPhie P. Miles E.W. J. Biol. Chem. 1993; 268: 8727-8734Abstract Full Text PDF PubMed Google Scholar, 6Rege V.D. Kredich N.M. Tai C.-H. Karsten W.E. Schnackerz K.D. Cook P.F. Biochemistry. 1996; 35: 13485-13493Crossref PubMed Scopus (38) Google Scholar, 7Watababe A. Kurokawa 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, 8Osterman A.L. Brooks H.B. Jackson L. Abbott J.J. Phillips M.A. Biochemistry. 1999; 38: 11814-11826Crossref PubMed Scopus (44) Google Scholar, 9Schirch D. Delle Fratte S. Iurescia S. Angelaccio S. Contestabile R. Bossa F. Schirch V. J. Biol. Chem. 1993; 268: 23132-23138Abstract Full Text PDF PubMed Google Scholar). In addition, lysine residues have also been shown to be involved in catalysis (4Nishimura K. Tanizawa K. Yoshimura T. Esaki N. Futaki S. Manning J.M. Soda K. Biochemistry. 1991; 30: 4072-4077Crossref PubMed Scopus (35) Google Scholar, 5Lu Z. Nagata S. McPhie P. Miles E.W. J. Biol. Chem. 1993; 268: 8727-8734Abstract Full Text PDF PubMed Google Scholar, 6Rege V.D. Kredich N.M. Tai C.-H. Karsten W.E. Schnackerz K.D. Cook P.F. Biochemistry. 1996; 35: 13485-13493Crossref PubMed Scopus (38) Google Scholar, 7Watababe A. Kurokawa 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, 10Toney M.D. Kirsch J.F. Science. 1989; 243: 1485-1488Crossref PubMed Scopus (215) Google Scholar, 11Ferreira G.C. Vajapey U. Hafez O. Hunter G.A. Barber M.J. Protein Sci. 1995; 4: 1001-1006PubMed Google Scholar), the formation of enzyme-substrate intermediates and product release (5Lu Z. Nagata S. McPhie P. Miles E.W. J. Biol. Chem. 1993; 268: 8727-8734Abstract Full Text PDF PubMed Google Scholar, 8Osterman A.L. Brooks H.B. Jackson L. Abbott J.J. Phillips M.A. Biochemistry. 1999; 38: 11814-11826Crossref PubMed Scopus (44) Google Scholar, 9Schirch D. Delle Fratte S. Iurescia S. Angelaccio S. Contestabile R. Bossa F. Schirch V. J. Biol. Chem. 1993; 268: 23132-23138Abstract Full Text PDF PubMed Google Scholar), and cofactor binding (4Nishimura K. Tanizawa K. Yoshimura T. Esaki N. Futaki S. Manning J.M. Soda K. Biochemistry. 1991; 30: 4072-4077Crossref PubMed Scopus (35) Google Scholar, 5Lu Z. Nagata S. McPhie P. Miles E.W. J. Biol. Chem. 1993; 268: 8727-8734Abstract Full Text PDF PubMed Google Scholar, 12Ferreira G.C. Neame P.J. Dailey H.A. Protein Sci. 1993; 2: 1959-1965Crossref PubMed Scopus (63) Google Scholar). Although progress in the spectroscopic and kinetic features of T. denticola cystalysin has been made recently, the catalytic mechanism and the individual residues essential for enzyme activity remain to be elucidated. To understand the functional contribution of the active site lysine residue (Lys-238) to stages of the reaction catalyzed by cystalysin, we have changed Lys-238 of cystalysin to Ala or Arg and have studied the kinetic and spectroscopic properties of the mutants. Evidence is provided that Lys-238, in addition to increasing the PLP binding affinity and facilitating the formation of external aldimine, plays an essential catalytic role in α,β-elimination. Materials—PLP, pyridoxamine 5′-phosphate (PMP), l-cystine, β-chloro-l-alanine, glycine, l-homoserine, l-methionine, NADH, pyruvate, rabbit muscle l-lactic dehydrogenase, methylamine, ethanolamine, ammonium chloride, and isopropyl β-d-thiogalactoside were from Sigma. l-cysteine and l-serine were from Fluka. All other chemicals were of highest grade commercially available. Site-directed Mutagenesis—The K238A and K238R mutant forms of cystalysin were made on the wild-type construct pUC18:hly (13Cordes E.H. Jencks W.P. Biochemistry. 1962; 1: 773-778Crossref PubMed Scopus (74) Google Scholar) using the QuikChange™ site-directed mutagenesis kit from Stratagene (La Jolla, CA). The kit employs double-stranded DNA as a 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 K238A and K238R mutants were produced using as primers 5′-GCTCCGTCTGCAACATTTAATATAGCAGGAATGGGC-3′, 5′-GCTCCGTCTAGAACATTTAATATAGCAGGAATGGGC-3′, and their complementary oligonucleotides, respectively. The coding regions of the mutated hly genes were sequenced to confirm the mutations. E. coli strain DH5α cells were transformed and used for expression. Expression and Purification of K238A and K238R Mutants—The conditions used for expression of the K238A and K238R mutants 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 (40) Google Scholar). The wild-type and mutant forms of cystalysin were purified to homogeneity with the following modifications of the procedure described previously (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). After the DEAE-FF-Sepharose XK 26/60 step, the enzymatic solution was concentrated to ∼6 ml and rechromatographed on the same resin (1.6 × 13-cm column) equilibrated in 10 mm potassium phosphate buffer, pH 7.6. The enzyme was eluted with a 225-ml linear gradient whose final concentration of potassium phosphate was 50 mm, pH 7.6. Active fractions were pooled and concentrated to ∼3 ml by Centriplus (Amicon). The protein concentration in all cystalysin samples was determined by absorbance spectroscopy using a previously determined extinction coefficient of 12.77 × 104m–1 cm–1 at 281 nm (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). PLP content of wild-type and mutant enzymes was determined by releasing the coenzyme in 0.1 m NaOH and using ϵ = 6600 m–1 cm–1 at 388 nm. Preparation and Reconstitution of Apo-K238A and Apo-K238R Mutants—Apoenzymes K238A and K238R were prepared by incubation of enzyme (∼1 mg/ml) with 0.5 m hydroxylamine in 0.5 m phosphate buffer, pH 6.9, for 3 h at 25 °C; this was followed by gel filtration on a desalting Hi-Prep column 26/10 equilibrated with 0.5 m phosphate buffer, pH 6.9. For reconstitution, a 10-fold molar excess of PLP was added, and, after 1 h, the solution was loaded on the above column previously equilibrated with 20 mm phosphate buffer, pH 7.4. The enzyme was then concentrated in microconcentrators. The apparent equilibrium constant for dissociation of PLP from K238A, Kd , was determined by measuring the molar fraction of enzyme-bound PLP, calculated as ([PLP]total – [PLP]free)/[K238A]total in the presence of PLP at a concentration varying from 20 to 950 nm. [PLP]total and [PLP]free were experimentally determined by HPLC in combination with ultrafiltration as described previously (14Bertoldi M. Borri Voltattorni C. Biochem. J. 2000; 352: 533-538Crossref PubMed Scopus (37) Google Scholar). The Kd value for dissociation of PLP from K238R was determined by measuring enzyme activity of the apoenzyme (2.7 μm) in the presence of PLP ranging from 0.05 to 20 μm. Enzyme Activity Assay—α,β-eliminase activity was measured by an spectrophotometric assay coupled with lactic dehydrogenase as reported previously (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). To determine the kinetic parameters of the catalysis, the assays were performed as indicated above using a fixed amount of enzyme, whereas l-cysteine or β-chloro-l-alanine concentration was varied from 0.15 to 10 mm. The experimental data were fit into the Michaelis-Menten equation to determine Km and k cat values. Spectrophotometric 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 the small amount of precipitate. Fluorescence spectra were taken with a FP750 Jasco spectrofluorometer using 5-nm excitation and emission bandwidths at a protein concentration varying from 1 to 8 μm. Spectra of blanks, i.e. samples containing all components except cystalysin, were taken immediately before the measurements of samples containing protein. Blank spectra were subtracted from the spectra containing the enzyme. CD spectra were obtained using a Jasco V-710 spectrophotometer with a thermostatically controlled compartment at 25 °C. For near-UV and visible wavelengths, protein concentration varied from 0.8 to 1 mg/ml in a cuvette with a 1-cm path length. Routinely, three spectra were recorded at a scan speed of 50 nm/min with a bandwidth of 2 nm and averaged automatically, except where indicated. For far-UV measurements, the protein concentration was 0.1 mg/ml with a 0.1-cm path length. Pre-steady-state Kinetic Analysis by UV-Vis Stopped-flow Spectroscopy—Wild-type cystalysin (7 μm) was mixed with glycine in 20 mm potassium phosphate, pH 7.4. Reactions proceeded at 25 °C, and coenzyme absorbance changes were monitored using a Biologic SFM3 mixer with a TC-100 (1-cm path length) quartz cell coupled to a BioKine PMS-60. The dead time was 3.6 ms at a flow velocity of 12 ml/s. Absorbance scans (500) from 300 to 550 nm were collected on a logarithmic time scale with a J&M Tidas 16 256 diode array detector (Molecular Kinetics, Pullman, WA) and analyzed using either SPECFIT (Spectrum Software Associates, Chapel Hill, NC) or Biokine 4.01 (Biologic, Claix, France) to determine the observed rate constants. Curve-fitting Analysis—Binding of ligands (L) to wild-type and active site lysine mutants was monitored by pre-steady-state and steady-state analysis, respectively. The data were fitted to a single exponential process (Equation 1, shown below) to obtain k obs. The binding of ligands to cystalysin, either wild-type or mutant, was followed by measuring the change in the absorbance and/or emission fluorescence intensity with time upon the addition of ligand. The kinetic measurements were performed at several ligand concentrations using an excess of ligand over enzyme concentration. For each ligand concentration, the time course was recorded, and the absorbance and/or fluorescence readings at each ligand concentration were fitted to a pseudo-first-order kinetic model, shown here in Equation 1, At=Aeq-(Aeq-A0)e-kobst(Eq. 1) where A 0 is the absorbance and/or emission fluorescence reading prior to ligand addition, At is the reading at the time t, and A eq is the reading at equilibrium For ligands where the dependence of k obs was linear, the data were fitted to Equation 2, shown here, x0026;x0026;E+L⇌koffkonELx0026;x0026;kobs=kon[L]+koff(Eq. 2) which describes a single-step binding model to determine k on and k off. k on is the apparent second-order rate constant for the formation of the Schiff base from free enzyme and ligand, and k off is the first-order rate constant for the decay of the Schiff base species to free enzyme and ligand. For ligands where k obs had a hyperbolic dependence on ligand concentration, the k obs data were fitted to Equation 3, shown here, x0026;x0026;E+L⇌KEL'EL'⇌k-2k+2ELx0026;x0026;kobs=k+2[L]KEL'+[L]+k-2(Eq. 3) which describes a two-step binding model, assuming that the first step is rapid, wherein KEL′ is the dissociation constant for the intermediate formed prior to the formation of the Schiff base species, and k +2 and k –2 are first-order rate constants for the interconversion between the intermediate (EL′) and the final Schiff base species (EL). The parameters in Equation 3 are related to those in Equation 2 as follows: k on = k +2/KEL′ and k off = k –2. Amplitude data (ΔA = A eq-A 0) were fitted to Equation 4, as shown, ΔA=Δε420nm[E][Gly](Kd+[Gly])(Eq. 4) to determine the dissociation constant (Kd) for Schiff base formation between glycine and wild-type cystalysin, where [E] represents the total concentration of the enzyme. As shown in Equation 5, Y=Ymax[E]t+[PLP]t+Kd-([E]t+[PLP]t+Kd)2-4[E]t[PLP]t2[E]t(Eq. 5) the Kd value of the mutant-coenzyme complex was obtained using a tight binding hypothesis. All data analysis to determine model-derived kinetic parameters was performed by nonlinear curve fitting using KaleidaGraph 3.52 (Synergy Software, Reading, PA). To define the functional role(s) of the lysine residue that forms an internal aldimine with PLP in the active site of cystalysin, we compared some spectroscopic and catalytic properties of the wild-type enzyme and mutants in which Lys-238 is replaced by alanine or arginine. The yield of K238A and K238R mutants after purification was ∼70–80% that of the wild-type protein. The purified mutant proteins were homogeneous, as indicated by a single band on SDS/PAGE. Spectroscopic Properties of K238A and K238R Mutants— Wild-type cystalysin displays characteristic absorption spectra with maxima at 418 and 320 nm, attributable to the ketoenamine form of the Schiff base and a substituted aldamine, respectively (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). The spectral properties of the PLP aldehyde are known to differ significantly from the internal aldimine and to give rise to maxima at 290, 390, and/or 335 nm (15Harris C.M. Johnson R.J. Metzler D.E. Biochim. Biophys. Acta. 1976; 421: 181-194Crossref PubMed Scopus (101) Google Scholar). As isolated, the K238A mutant displays an absorption maximum at 412 nm, whereas the K238R mutant exhibits two peaks, one centered at 390–400 nm and the other at 320 nm. K238A and K238R mutants bind 1 and 0.37 mol of PLP per mol of dimer, respectively. The ratio A 320/A 390–400 in the K238R mutant is ∼3. HPLC analysis of the supernatants, obtained after reduction of the mutants with NaBH4 followed by denaturation and centrifugation, indicates the presence of two peaks. On the basis of its retention time, one peak, corresponding to ∼43 and 78% of the original coenzyme content of K238A and K238R mutants, respectively, was identified as pyridoxine 5′-phosphate (PNP). The other peak does not migrate with the same retention time as any of the controls (PLP, PMP, PNP) but elutes with a retention time similar to those of PLP adducts with amine or amino acids and most likely arises from the adduct of PLP with an uncharacterized compound. The reconstituted holoenzymes K238A and K238R (obtained by complete removal of the cofactor followed by treatment with 10-fold molar excess of PLP and removal of unbound coenzyme (described under “Experimental Procedures”)) contained 1.0 and 1.1 mol of PLP per dimer, respectively, as judged by NaOH treatment and HPLC analysis. As shown in Fig. 1A, whereas the reconstituted K238A mutant exhibits an absorption band at 400 nm, the reconstituted K238R mutant shows two absorbance maxima at 390 and 320 nm. Absorption bands at 388 and 330 nm are present in the spectra of free PLP. However, in PLP free in solution, the 388-nm chromophore is the most abundant species, whereas it is the minor component of the spectrum of K238R mutant. The ratio of A 280/A 400 is 15 for the reconstituted K238A mutant compared with an A 280/A 418 value of 10 for wild-type cystalysin; for reconstituted mutant K238R, the A 280/A 390 and A 280/A 320 ratios are ∼40 and 15, respectively. After reduction of the reconstituted mutants with sodium borohydride followed by denaturation with trichloroacetic acid, HPLC analysis of the supernatant reveals that the original PLP content was completely converted into PNP. When the wild-type enzyme was treated with NaCNBH3, which does not readily reduce a free aldehyde group but does reduce a Schiff base (16Lane C.F. Synthesis. 1975; 3: 135-146Crossref Scopus (547) Google Scholar), the absorption at 418 nm was diminished in parallel with an increase in the absorption at 330 nm. This result is consistent with reduction of the internal Schiff base formed between the aldehyde group of PLP and the ϵ-amino group of Lys-238 in the wild type enzyme. In contrast, treatment of both reconstituted mutants with NaCNBH3 did not alter the spectra. Altogether, these data indicate that reconstitution of both mutants with PLP gives rise to proteins in which PLP binds as the free aldehyde. Thus, these reconstituted forms of K238A and K238R mutants were used for the following kinetic and spectral analysis. The absorption spectra of the internal aldimine of the wild-type depend on pH; titration of the enzyme-bound absorbance over the pH range 6–9.7 has shown that the 320-nm band increases at high pH values, whereas the 418-nm band decreases (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). In contrast, no changes in absorption spectra of apomutants K238A and K238R reconstituted with PLP were observed between pH 6 and 9.7. Fig. 1B shows CD spectra of wild-type and reconstituted K238A and K238R enzymes in the visible and near-UV region. PLP bound to the wild-type exhibited a positive circular dichroism band at 412 nm, as reported previously (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). In contrast, whereas the K238A mutant shows a negative Cotton effect with λmax at 407 nm and a molar ellipticity lower than that of the positive Cotton effect of the wild-type enzyme, the K238R mutant displays two modest signals, a negative band in the 400-nm region and a positive dichroic signal at 320 nm. With respect to wild-type, both mutants also display a decrease of negative dichroic bands in the aromatic region of 288–296 nm and an increase of the positive band at 275 nm. Spectra in the near-UV region of both mutants are reminiscent of that of wild-type apocystalysin, as shown in Fig. 1B. The circular dichroism spectra in the range 190–240 nm of both mutants and the wild-type enzyme appeared almost indistinguishable; they display minima at 222 and 208 nm, which are characteristic of a protein with a high content of α-helical structure (data not shown). These data suggest that, although small conformational changes may occur in the region immediately surrounding the site of the mutation, no appreciable change in the secondary or tertiary structure occurs upon the substitution of alanine or arginine for Lys-238. When excited at 281 nm, the fluorescence emission spectrum of wild-type cystalysin shows two maxima at 337 nm and ∼500 nm; the ratio of F 337/F 500 is ∼86 (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). The band at the shorter wavelength is a result of intrinsic tryptophan fluorescence, whereas the band at the longer wavelength is due to the delayed Schiff base fluorescence. Excitation at 281 nm of both reconstituted K238A and K238R mutants shows an emission spectrum with a maximum at only 337 nm whose intensity is 1.5–1.6-fold higher than that of the wild-type. Excitation of the wild-type enzyme at 418 nm results in a faint emission band at 504 nm (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). Upon excitation at 400 nm, whereas reconstituted K238A mutant emitted at 499 nm with an emission intensity 2-fold higher that that of the wild-type, reconstituted K238R shows no emission at ∼500 nm. Excitation at 320 nm of reconstituted K238R mutant shows an ∼3-fold increased emission fluorescence intensity when compared with that of the wild-type with a maximum at 371 nm (red-shifted 4 nm with respect to the maximum of the wild-type) (Fig. 1C). Binding Affinity of K238A and K238R Mutants for PLP Cofactor—The data for reconstitution to K238A or K238R holoenzymes versus PLP concentration have been collected by measuring the molar fraction of enzyme-bound PLP or the recovery of enzyme activity, respectively (see “Experimental Procedures”). Titration analyses of both apomutants with PLP fitted to Equation 5 yielded Kd values for PLP-K238A and PLP-K238R complexes equal to 70 ± 16 nm and 0.9 ± 0.1 μm, respectively (data not shown). The apparent Kd for dissociation of PLP from wild-type has been found to be 6.6 ± 1nm (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). Thus, substitution by alanine or arginine, in addition to resulting in a significant loss of the bound coenzyme, apparently causes a ∼ 10- and 150-fold decrease in PLP-binding affinity, respectively. Kinetic Properties of K238A and K238R Mutants—Apomutant K238A, reconstituted with PLP, was essentially inactive under the standard assay conditions in which the k cat values for the wild-type toward l-cysteine or β-chloro-l-alanine were ∼11 s–1 or 60 s–1 at 25 °C, respectively (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). On the contrary, the reconstituted K238R mutant displays a detectable eliminase activity. Steady-state kinetics data for the reaction of this mutant were collected for a range of l-cysteine or β-chloro-l-alanine concentrations by measurement of pyruvate formation. Although the Km values (0.6 ± 0.1 mm for l-cysteine, 1.9 ± 0.2 mm for β-chloro-l-alanine) are not significantly altered by mutation, the k cat values for the reaction are 1.12 ± 0.05 min–1 for l-cysteine and 6.8 ± 0.3 min–1 for β-chloro-l-alanine, ∼ 500-fold lower than that for the wild-type (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). Thus, per mole of cofactor, the k cat of this mutant is 0.3% of that of the wild-type. Formation of External Aldimine—Glycine behaves as a non-productive analogue of wild-type cystalysin. The addition of glycine to cystalysin causes the immediate appearance of an absorption band at 429 nm attributable to the formation of the external aldimine (2Bertoldi M. Cellini B. Clausen T. Borri Voltattorni C. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (40) Google Scholar). Thus, the interaction between cystalysin and glycine represents a useful model for the analysis of formation and hydrolysis of the external Schiff base, and the comparison between the reactions of wild-type and Lys-238 mutants with this ligand could provide insight into the role of active site lysine in a step that is on the catalytic pathway of cystalysin. The kinetics of glycine binding to wild-type cystalysin is too rapid to be measured with a conventional spectrophotometer. Thus, the reaction of wild-type with glycine was followed by multiwavelength (300–550 nm) stopped-flow spectroscopy under pre-steady-state conditions. Upon mixing cystalysin with glycine, the spectrum of the initial species is similar to that of free enzyme, except that the absorption maximum at 418 nm is red-shifted 11 nm, indicating that a fast process is complete within the dead t