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Multifunctional Role of His159in the Catalytic Reaction of Serine Palmitoyltransferase

2009; Elsevier BV; Volume: 284; Issue: 23 Linguagem: Inglês

10.1074/jbc.m808916200

1083-351X

Y Shiraiwa, Hiroko Ikushiro, Hideyuki Hayashi,

Polyamine Metabolism and Applications

Serine palmitoyltransferase (SPT) belongs to the fold type I family of the pyridoxal 5′-phosphate (PLP)-dependent enzyme and forms 3-ketodihydrosphingosine (KDS) from l-serine and palmitoyl-CoA. Like other α-oxamine synthase subfamily enzymes, SPT is different from most of the fold type I enzymes in that its re face of the PLP-Lys aldimine is occupied by a His residue (His159) instead of an aromatic amino acid residue. His159 was changed into alanine or aromatic amino acid residues to examine its role during catalysis. All mutant SPTs formed the PLP-l-serine aldimine with dissociation constants several 10-fold higher than that of the wild type SPT and catalyzed the abortive transamination of l-serine. These results indicate that His159 is not only the anchoring site for l-serine but regulates the α-deprotonation of l-serine by fixing the conformation of the PLP-l-serine aldimine to prevent unwanted side reactions. Only H159A SPT retained activity and showed a prominent 505-nm absorption band of the quinonoid species during catalysis. Global analysis of the time-resolved spectra suggested the presence of the two quinonoid intermediates, the first formed from the PLP-l-serine aldimine and the second from the PLP-KDS aldimine. Accumulation of these quinonoid intermediates indicated that His159 promotes both the Claisen-type condensation as an acid catalyst and the protonation at Cα of the second quinonoid to form the PLP-KDS aldimine. These results, combined with the previous model building study (Ikushiro, H., Fujii, S., Shiraiwa, Y., and Hayashi, H. (2008) J. Biol. Chem. 283, 7542–7553), lead us to propose a novel mechanism, in which His159 plays multiple roles by exploiting the stereochemistry of Dunathan's conjecture. Serine palmitoyltransferase (SPT) belongs to the fold type I family of the pyridoxal 5′-phosphate (PLP)-dependent enzyme and forms 3-ketodihydrosphingosine (KDS) from l-serine and palmitoyl-CoA. Like other α-oxamine synthase subfamily enzymes, SPT is different from most of the fold type I enzymes in that its re face of the PLP-Lys aldimine is occupied by a His residue (His159) instead of an aromatic amino acid residue. His159 was changed into alanine or aromatic amino acid residues to examine its role during catalysis. All mutant SPTs formed the PLP-l-serine aldimine with dissociation constants several 10-fold higher than that of the wild type SPT and catalyzed the abortive transamination of l-serine. These results indicate that His159 is not only the anchoring site for l-serine but regulates the α-deprotonation of l-serine by fixing the conformation of the PLP-l-serine aldimine to prevent unwanted side reactions. Only H159A SPT retained activity and showed a prominent 505-nm absorption band of the quinonoid species during catalysis. Global analysis of the time-resolved spectra suggested the presence of the two quinonoid intermediates, the first formed from the PLP-l-serine aldimine and the second from the PLP-KDS aldimine. Accumulation of these quinonoid intermediates indicated that His159 promotes both the Claisen-type condensation as an acid catalyst and the protonation at Cα of the second quinonoid to form the PLP-KDS aldimine. These results, combined with the previous model building study (Ikushiro, H., Fujii, S., Shiraiwa, Y., and Hayashi, H. (2008) J. Biol. Chem. 283, 7542–7553), lead us to propose a novel mechanism, in which His159 plays multiple roles by exploiting the stereochemistry of Dunathan's conjecture. Coenzymes act as catalysts in biological systems, and many enzymes require coenzymes as the important catalytic group. In most cases, coenzymes can carry out the catalysis in the absence of the enzyme protein. However, the reaction rate is much lower than the rate in the system containing the enzyme protein. Furthermore, the reaction specificity is reduced in the nonenzymatic system; coenzymes without the enzyme protein tend to undergo side reactions. A remarkable example is the coenzyme pyridoxal 5′-phosphate (PLP). 3The abbreviations used are:PLPpyridoxal 5′-phosphatePMPpyridoxamine 5'-phosphateHPLChigh performance liquid chromatographyKDS3-ketodihydrosphingosineSPTserine palmitoyltransferaseWTwild type. 3The abbreviations used are:PLPpyridoxal 5′-phosphatePMPpyridoxamine 5'-phosphateHPLChigh performance liquid chromatographyKDS3-ketodihydrosphingosineSPTserine palmitoyltransferaseWTwild type. PLP is a versatile catalyst catalyzing transamination, decarboxylation, elimination, aldol cleavage, Claisen-type condensation, etc. of amino acids. Therefore, a pyridoxal enzyme is required to have a structure that enables elaborated chemical mechanism by which only a specific reaction proceeds at each catalytic step. pyridoxal 5′-phosphate pyridoxamine 5'-phosphate high performance liquid chromatography 3-ketodihydrosphingosine serine palmitoyltransferase wild type. pyridoxal 5′-phosphate pyridoxamine 5'-phosphate high performance liquid chromatography 3-ketodihydrosphingosine serine palmitoyltransferase wild type. Serine palmitoyltransferase (SPT) catalyzes the condensation reaction of l-serine and palmitoyl-CoA to produce 3-ketodihydrosphingosine (KDS) (1.Hanada K. Biochim. Biophys. Acta. 2003; 1632: 16-30Crossref PubMed Scopus (454) Google Scholar). This is the first step in the sphingolipid biosynthesis. SPT belongs to the PLP-dependent α-oxamine synthase subfamily containing 5-aminolevulinate synthase, 8-amino-7-oxononanoate synthase, and 2-amino-3-ketobutyrate CoA ligase (2.Hayashi H. J. Biochem. (Tokyo). 1995; 118: 463-473Crossref PubMed Scopus (150) Google Scholar, 3.Mehta P.K. Christen P. Adv. Enzymol. Rel. Areas Mol. Biol. 2000; 74: 129-184PubMed Google Scholar, 4.Salzmann D. Christen P. Mehta P.K. Sandmeier E. Biochem. Biophys. Res. Commun. 2000; 270: 576-580Crossref PubMed Scopus (15) Google Scholar, 5.Schneider G. Kack H. Lindqvist Y. Structure. 2000; 8: R1-R6Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 6.Eliot A.C. Kirsch J.F. Annu. Rev. Biochem. 2004; 73: 383-415Crossref PubMed Scopus (658) Google Scholar). All of them have been successfully crystallized, and their three-dimensional structures have been determined (7.Alexeev D. Alexeeva M. Baxter R.L. Campopiano D.J. Webster S.P. Sawyer L. J. Mol. Biol. 1998; 284: 401-419Crossref PubMed Scopus (108) Google Scholar, 8.Hunter G.A. Ferreira G.C. Biochemistry. 1999; 38: 3711-3718Crossref PubMed Scopus (35) Google Scholar, 9.Webster S.P. Alexeev D. Campopiano D.J. Watt R.M. Alexeeva M. Sawyer L. Baxter R.L. Biochemistry. 2000; 39: 516-528Crossref PubMed Scopus (110) Google Scholar, 10.Schmidt 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, 11.Astner I. Schulze J.O. van den Heuvel J. Jahn D. Schubert W.D. Heinz D.W. EMBO J. 2005; 24: 3166-3177Crossref PubMed Scopus (163) Google Scholar, 12.Yard B.A. Carter L.G. Johnson K.A. Overton I.M. Dorward M. Liu H. McMahon S.A. Oke M. Puech D. Barton G.J. Naismith J.H. Campopiano D.J. J. Mol. Biol. 2007; 370: 870-886Crossref PubMed Scopus (97) Google Scholar). These enzymes belong to the fold type I family of the PLP-dependent enzymes according to their folding pattern (5.Schneider G. Kack H. Lindqvist Y. Structure. 2000; 8: R1-R6Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 6.Eliot A.C. Kirsch J.F. Annu. Rev. Biochem. 2004; 73: 383-415Crossref PubMed Scopus (658) Google Scholar). The commonly known fold type I PLP-dependent enzymes have an aromatic amino acid residue locating at the re face of the PLP-Lys internal aldimine and stacking with the pyridine ring of PLP. On the other hand, all members of the PLP-dependent α-oxamine synthase subfamily known to date have a His residue in this position. Therefore, the His residue is expected to play unique roles in the reaction mechanism of the PLP-dependent α-oxamine synthase subfamily enzymes. Scheme 1 shows the chemical reaction mechanism of SPT (1.Hanada K. Biochim. Biophys. Acta. 2003; 1632: 16-30Crossref PubMed Scopus (454) Google Scholar, 13.Ikushiro H. Fujii S. Shiraiwa Y. Hayashi H. J. Biol. Chem. 2008; 283: 7542-7553Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). At the active site of SPT, PLP forms an aldimine with the ϵ-amino group of Lys265 (internal aldimine, I). The internal aldimine undergoes transaldimination with the first substrate l-serine to yield the PLP-l-serine aldimine (external aldimine, II). After binding of the second substrate palmitoyl-CoA, α-deprotonation occurs to form the first quinonoid intermediate (III). The carbanionic Cα of III attacks palmitoyl-CoA (Claisen-type condensation) to generate a condensation product (IV), which, by decarboxylation, yields the second quinonoid intermediate (V). Protonation at Cα of V gives the external aldimine of PLP-KDS (VI). Finally, release of KDS regenerates the internal aldimine (I). For this reaction mechanism, we proposed by model building studies that His159 of SPT is the anchoring site for both l-serine and palmitoyl-CoA and possibly involved in the catalytic steps (13.Ikushiro H. Fujii S. Shiraiwa Y. Hayashi H. J. Biol. Chem. 2008; 283: 7542-7553Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). However, no experimental analyses have been made to confirm this proposal or to gain further insight into the function of the residue. To determine the catalytic role of His159, especially its role in the reaction specificity of PLP-dependent α-oxamine synthase subfamily enzymes, we constructed mutant Sphingomonas paucimobilis SPTs, in which His159 was replaced by Ala and aromatic amino acid residues, and analyzed the reaction of these mutant enzymes. The results showed that His159 has at least two additional distinct functions: one as a residue that controls the reaction pathway by adjusting the conformation of the PLP-l-serine external aldimine and the other as an acid catalyst that promotes the reactions of the Claisen-type condensation and the following steps. Palmitoyl-CoA was obtained from Funakoshi (Tokyo, Japan). Escherichia coli BL21 (DE3) pLysS and plasmid pET21b were from Novagen (Madison, WI). S-(2-Oxoheptadecyl)-CoA was synthesized as previously described (13.Ikushiro H. Fujii S. Shiraiwa Y. Hayashi H. J. Biol. Chem. 2008; 283: 7542-7553Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). 3-Keto-dihydrosphingosine hydrochloride was obtained from Matreya LLC (Pleasant Gap, PA). All other chemicals were of the highest grade commercially available. The mutation of His159 to Ala (H159A), Phe (H159F), Tyr (H159Y), and Trp (H159W) was introduced by a two-step PCR as previously described (13.Ikushiro H. Fujii S. Shiraiwa Y. Hayashi H. J. Biol. Chem. 2008; 283: 7542-7553Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). H159A was purified in the same way as the wild type enzyme (14.Ikushiro H. Hayashi H. Kagamiyama H. Biochemistry. 2004; 43: 1082-1092Crossref PubMed Scopus (58) Google Scholar). Other mutant proteins were purified accordingly except for the additional purification by DEAE-Sepharose Fast Flow and the Butyl-Sepharose Fast Flow chromatographies on an ÄKTA Protein Purification System (GE Healthcare Bio-Sciences AB). The buffer solution was 20 mm Tris-HCl buffer (pH 7.5). Usually, 80 mg of the mutant proteins were obtained from 1 liter of culture. All of the spectroscopic measurements were carried out in 50 mm HEPES-NaOH, 50 mm KCl, and 0.1 mm EDTA, pH 7.5, at 298 K. The absorption spectra were measured using a Hitachi U-3300 spectrophotometer (Tokyo, Japan). The concentration of the SPT subunit in solution was spectrophotometrically determined. The apparent molar extinction coefficient at 280 nm for the PLP form of SPT was 28,300 m−1 cm−1, which was calculated on the basis of the number of tryptophan and tyrosine residues in SPT (15.Ikushiro H. Hayashi H. Kagamiyama H. J. Biol. Chem. 2001; 276: 18249-18256Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Stopped flow spectrophotometry was performed using an Applied Photophysics (Leatherhead, UK) four-syringe SX.18MV spectrophotometer equipped for both conventional and sequential stopped flow measurements. The reactions were carried out in 50 mm HEPES-NaOH, 150 mm KCl, and 0.1 mm EDTA, pH 7.5, at 298 K. For the sequential stopped flow measurements, two solutions (enzyme and l-serine) were mixed and allowed to stand for an appropriate time in the aging loop. After a programmable delay of 1 s, when most of the PLP-l-serine external aldimine had been formed, the contents of the aging loop were mixed with the third solution, palmitoyl-CoA. The dead time was 2.3 ms under a gas pressure of 600 kPa. Time-resolved spectra were collected using the SX.18MV system equipped with a photodiode array accessory and the XScan (version 1.0) controlling software. The absorption changes were analyzed using the software Pro-KII (Applied Photophysics). The high performance liquid chromatography (HPLC) analysis was performed according to Ref. 16.Valls F. Sancho M.T. Fernandez-Muino M.A. Checa M.A. J. Agric. Food Chem. 2001; 49: 38-41Crossref PubMed Scopus (32) Google Scholar with minor modifications. H159A SPT was incubated in the HEPES buffer in the presence or absence of 200 mm l-serine at room temperature overnight, and then perchloric acid was added to a final concentration of 5% (v/v). The samples were centrifuged at 16,000 × g for 15 min, and the resulting supernatants were analyzed by HPLC using a prepacked C18 reversed phase column (Cosmosil 5C18-AR-II, 4.6 × 150 mm; Nacalai Tesque, Kyoto, Japan). The mobile phase was 50 mm potassium phosphate, pH 3.2, acetonitrile (99/1, v/v), and the flow rate was 1 ml/min. PLP and PMP were detected by fluorescence at 395 nm after excitation at 290 nm. The synthetic strategy for the preparation of [α-2H]l-serine is based on the fact that deprotonation at Cα of the l-serine at the active site of SPT is accelerated by the presence of S-(2-oxoheptadecyl)-CoA, an analogue of palmitoyl-CoA (13.Ikushiro H. Fujii S. Shiraiwa Y. Hayashi H. J. Biol. Chem. 2008; 283: 7542-7553Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). A mixture containing 100 mm l-serine, 100 μm S-(2-oxoheptadecyl)-CoA, and 40 μm SPT were incubated overnight at room temperature in 50 mm potassium pyrophosphate buffer (D2O, pH 7.5). The α-proton peak in the 1H NMR spectrum disappeared after 24 h (data not shown). At this point, SPT was removed from the reaction mixture by ultrafiltration using VivaspinTM (Sartorius Stedim Biotech, Aubagne, France). The filtrate solution was adjusted to pH 3 with HCl and applied to a Dowex-50 column. [α-2H]l-Serine was eluted with 1 m NH4OH and collected. It was then dried and recrystallized. The yield was 72.8%. The SPT activity was measured using [14C]l-serine as previously described (13.Ikushiro H. Fujii S. Shiraiwa Y. Hayashi H. J. Biol. Chem. 2008; 283: 7542-7553Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Briefly, 10 μm SPT mutants were incubated with [14C]l-serine and palmitoyl-CoA in 50 mm HEPES-NaOH buffer (pH 7.5) containing 0.2 or 1 mm PLP for 10 min at 298 K. The radiolabeled product KDS was separated by thin layer chromatography and quantified. The apoenzymes of several mutant SPTs were prepared by removing PLP as phenylhydrazone (17.Hayashi H. Mizuguchi H. Kagamiyama H. Biochemistry. 1993; 32: 812-818Crossref PubMed Scopus (74) Google Scholar). H159A SPT was obtained by column chromatography as an apoenzyme. Therefore, titration of the apoenzyme with PLP was performed to estimate the PLP binding ability. When PLP was added to the H159A SPT enzyme solution, new absorption bands appeared at 332 and 412 nm (Fig. 1A), indicating the formation of the PLP-Lys265 aldimine at the active site of the enzyme. Plots of A430 versus [PLP] showed a biphasic increase in A430 with a transition point at around the [PLP] equal to the enzyme concentration (28 μm), showing that PLP is bound 1:1 to the enzyme subunit (Fig. 1B). A430 is therefore expressed as follows, A430=12∈hole−∈PLP×Kd+Et+PLPt−Kd+Et+PLPt2−4EtPLPt+∈PLPPLPt(Eq. 1) where ϵPLP and ϵholo denote the absorptivity at 430 nm of the free PLP and the holoenzyme, respectively, and Kd is the dissociation constant for the enzyme and PLP. A theoretical line based on Equation 1 was fitted to the experimental values (Fig. 1B), and the parameters were determined to be ϵPLP = 1320 ± 3 m−1 cm−1, ϵholo = 2840 ± 70 m−1 cm−1, and Kd = 18.9 ± 3.0 μm. When H159A SPT saturated with PLP was subjected to gel filtration to remove the excess PLP, the enzyme essentially showed the same spectra as WT SPT (Fig. 1A, dashed line) with two absorption bands at 332 and 412 nm, each corresponding to the enolimine and ketoenamine forms of the internal aldimine. Contrary to H159A SPT, the purified H159F, H159Y, and H159W SPTs were not complete apoenzymes, and the apoenzymes of these mutant SPTs were obtained by treatment with phenylhydrazine. In the spectra of these mutant enzymes saturated with PLP, the intensity of the 412-nm absorption band was greater than that in H159A SPT (data not shown). This increase in intensity was considered to occur because the ketoenamine form was stabilized in H159F, H159Y, and H159W SPTs. Titration with PLP was carried out in the same way as for H159A SPT. The apoenzyme of WT SPT was also treated with phenylhydrazine and titrated with PLP. The apparent dissociation constant (Kd) is summarized in Table 1. The value of Kd of the mutant SPTs was lower than that of WT. This indicates that His159 does not play significant roles in the binding of PLP to the enzyme.TABLE 1Interactions of WT and mutant of SPTs with PLP andl-serineEnzymesKd for PLPKd forl-serinek for abortive transaminationμmmms−1WT27.3 ± 2.61.4 ± 0.1NDH159A18.9 ± 3.077.1 ± 14.4(4.43 ± 0.02) × 10−4H159F3.0 ± 0.427.8 ± 2.0(11.67 ± 0.03) × 10−5H159Y12.9 ± 3.619.8 ± 1.5(6.34 ± 0.04) × 10−5H159W17.3 ± 3.121.6 ± 1.5(6.75 ± 0.03) × 10−5 Open table in a new tab When [14C]l-serine was used as the substrate, the radiolabeled product KDS was detected only in H159A among the mutant SPTs (Fig. 2). Therefore, steady state kinetic analysis of H159A SPT was performed. When PLP was not added to the reaction mixture, the rate of product formation by H159A SPT rapidly declined with time. On the other hand, the time course was almost linear up to 20 min in the presence of 0.2 mm PLP, provided that the l-serine concentration was below 20 mm (data not shown). At higher concentrations of l-serine, it was necessary to increase the concentration of PLP to obtain the linear time course up to 20 min. These nonlinear behaviors are due to the abortive transamination catalyzed by H159A (see the next section), which becomes pronounced with increasing substrate concentration. The rate for the production of KDS was plotted versus the substrate concentrations (supplemental Fig. S1), and the theoretical lines based on the steady state ordered Bi-Bi mechanism were fitted to the experimental values. The kinetic parameters were obtained under two different conditions: condition A (0.2 mm PLP, 2–20 mm l-serine, and 0.01–2 mm palmitoyl-CoA), Km (l-serine) = 128 ± 51 mm, Km (palmitoyl-CoA) = 1.6 ± 0.7 mm, and kcat = 0.200 ± 0.070 s−1; and condition B (1 mm PLP, 2–200 mm l-serine, and 0.01–5 mm palmitoyl-CoA), Km (l-serine) = 58.1 ± 5.5 mm, Km (palmitoyl-CoA) = 0.72 ± 0.08 mm, and kcat = 0.112 ± 0.004 s−1. The corresponding values of WT SPT are Km (l-serine) = 6.2 ± 0.6 mm, Km (palmitoyl-CoA) = 1.0 ± 0.1 mm, and kcat = 0.69 ± 0.03 s−1, which were obtained under the condition of 10 μm PLP, 2–20 mm l-serine, and 0.01–2 mm palmitoyl-CoA (13.Ikushiro H. Fujii S. Shiraiwa Y. Hayashi H. J. Biol. Chem. 2008; 283: 7542-7553Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Unfortunately, neither of the two conditions is ideal for obtaining the kinetic parameters of H159A SPT; condition A uses the l-serine concentrations lower than the Km value, and condition B uses a high concentration of PLP, which tends to undergo nonspecific modification of Lys residues of enzyme proteins and may alter their properties (18.Hädener A. Alefounder P.R. Hart G.J. Abell C. Battersby A.R. Biochem. J. 1990; 271: 487-491Crossref PubMed Scopus (15) Google Scholar). However, we can say that the Km for l-serine is apparently increased (20-fold under condition A and 10-fold under condition B) by the H159A mutation. The H159A mutation did not significantly decrease the kcat value; H159A SPT retained 29% (condition A) or 16% (condition B) of the activity of WT SPT. The Km for palmitoyl-CoA was also essentially unaffected by the H159A mutation. The addition of l-serine to H159A SPT caused an increase in the absorption band at 416 nm and a concomitant decrease in the absorption band at 332 nm (Fig. 3, line 1). As in the case of WT SPT, this is considered to reflect the formation of the external aldimine, in which the ketoenamine tautomer became dominant as compared with the internal aldimine. However, the spectrum of the external aldimine was not stable, and the intensity of the 416-nm absorption band gradually decreased, and a new absorption band appeared at 326 nm (Fig. 3). After the spectral transition was over, the enzyme solution was deproteinized and subjected to HPLC analysis. A stoichiometric amount of PMP was found to be formed from the PLP of the holoenzyme incubated with l-serine (supplemental Fig. S2). These results indicate that the external aldimine in H159A SPT undergoes an abortive transamination. The decrease in the absorption at 416 nm was fitted to an exponential curve, and the rate constant for the abortive transamination was calculated to be (4.43 ± 0.02) × 10−4 s−1. H159F, H159Y, and H159W SPT reacted with l-serine in a manner similar to H159A SPT, and the rate constants for the abortive transamination were obtained (Table 1). Because the external aldimine is not stable in the His159 mutant enzymes, the dependence of the formation of the external aldimine with l-serine was followed by stopped flow spectrophotometry. The absorption at 416 nm exponentially increased and then gradually decreased upon mixing the enzymes with l-serine (data not shown). Therefore, the spectra at which A416 reached the maximum value (generally 1 s after mixing, depending on the l-serine concentration) were collected from each set of the time-resolved spectra. The absorbance at 416 nm was plotted versus the l-serine concentration, and the dissociation constant (Kd) was obtained in the same way as described in Ref. 14.Ikushiro H. Hayashi H. Kagamiyama H. Biochemistry. 2004; 43: 1082-1092Crossref PubMed Scopus (58) Google Scholar. The Kd value of H159A SPT was 77.1 mm and was 50-fold greater than that of WT SPT. The other mutant enzymes showed Kd values greater than that of WT SPT but severalfold lower than that of H159A SPT (Table 1). Among the His159 mutant SPTs, only H159A SPT showed any enzyme activity. Therefore, a transient kinetic analysis of the reaction of H159A SPT with l-serine and palmitoyl-CoA was carried out using a sequential stopped flow system. This system was necessary because the external aldimine complex of H159A SPT with l-serine must react with the palmitoyl-CoA before it undergoes abortive transamination. The enzyme solution (200 μm) was mixed with an equal volume of 400 mm l-serine and allowed to stand for 1 s in the aging loop, and then the enzyme-l-serine complex was mixed 1:1 with various concentrations of palmitoyl-CoA containing 200 mm l-serine. PMP formed during the 1 s was considered to be negligible as calculated from the rate constant of the abortive transamination. The time-resolved spectra showed a new absorption band at 505 nm, indicating the formation of the quinonoid intermediate (Fig. 4A). The singular value decomposition implemented to the Pro-KII software indicated that the spectral eigenvector suggests the presence of two spectroscopically distinct species. The simplest model that accounts for this is the following equation, A+PalCoA⇄kdB→k+3D⇄k−4k+4A+KDS(Eq. 2) where A and B denote the enzyme-l-serine complex and the enzyme-l-serine-palmitoyl-CoA ternary complex, respectively, and A and B are assumed to be in rapid equilibrium. Because the experiment was carried out in the presence of a nearly saturating concentration of l-serine, and the rate constant for the formation of the external aldimine is of the order of 1000 s−1 (14.Ikushiro H. Hayashi H. Kagamiyama H. Biochemistry. 2004; 43: 1082-1092Crossref PubMed Scopus (58) Google Scholar), the free enzyme generated from D by dissociating KDS was assumed to be rapidly converted to A (the symbol D was used here for the sake of consistency with the model of Equation 3). The global analysis based on this model yielded a quinonoid-like spectrum for the intermediate D (data not shown), but its absorptivity at 505 nm reached the value of 4.1 × 108 m−1 cm−1, which was 104-fold higher than that of the typical quinonoid intermediate (13.Ikushiro H. Fujii S. Shiraiwa Y. Hayashi H. J. Biol. Chem. 2008; 283: 7542-7553Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Additionally, the rate constant for B → D was obtained to be 2.6 × 10−5 s−1, which was far lower than the kcat value of H159A SPT. Therefore, the model of Equation 2 was considered to be inadequate for describing the results of Fig. 4A. We then added an intermediate C to the above model. A+PalCoA⇄kdB⇄k−2k+2C→k+3D⇄k−4k+4A+KDS(Eq. 3) The global analysis based on this model yielded the spectra of the intermediates and the values of the kinetic parameters, each shown in Fig. 4B and Table 2, respectively. Both C and D have absorption maximum at around 500 nm with molar absorptivity values above 30,000 m−1 cm−1. Thus, C and D are the quinonoid intermediates and are reasonably considered to correspond to III and V, respectively, in Scheme 1. The abortive transamination was not included in Equation 3. This is rationalized by the fact that k+2 = 0.46 s−1 was much greater than 4.4 × 10−4 s−1 of the rate constant of the abortive transamination. Considering the Kd value for l-serine (77 mm), H159A SPT is not completely saturated with 200 mm of l-serine. Therefore, although the A of Equation 3 is largely the external aldimine, it contains a small fraction of the internal aldimine. This, however, does not affect the kinetic parameters and the absorption spectra of the intermediates in Equation 3 except for Kd and the spectrum of A, both of which are irrelevant to the following discussion.TABLE 2Kinetic parameters obtained from the transient kinetic analysisSubstratel-Serine[α-2H]l-SerineKd (mm)0.0604 ± 0.00010.0924 ± 0.0002k+2(s−1)0.46 ± 0.200.064 ± 0.004K−2(s−1)3.48 ± 2.262.19 ± 0.49k+3(s−1)4.10 ± 2.423.50 ± 0.26k+4(s−1)3.30 ± 0.123.34 ± 0.19k−4(mm−1s−1)262 ± 21140 ± 37 Open table in a new tab A similar kinetic analysis was carried out for the reaction of H159A SPT with [α-2H]l-serine and palmitoyl-CoA, to detect the isotopically sensitive step. The time-resolved spectra are shown in Fig. 4C. Accumulation of the quinonoid intermediate was less than that observed for the reaction with l-serine and palmitoyl-CoA. The global analysis of the time-resolved spectra gave, however, intermediate spectra essentially identical to those obtained from the reaction with l-serine and palmitoyl-CoA (Fig. 4D). Among the kinetic parameters, only k+2 showed a high kinetic isotope effect: 0.064 ± 0.004 s−1 for [α-2H]l-serine compared with 0.46 s−1 for l-serine (Table 2). The kinetic isotope effect value of 7.2 is very close to the value of 7.3 for the 1,3-prototropic shift in aminotransferases (19.Onuffer J.J. Kirsch J.F. Prot. Eng. 1994; 7: 413-424Crossref PubMed Scopus (60) Google Scholar), which also involves deprotonation at Cα. This strongly indicates that k+2 really represents the α-deprotonation and supports that C in Equation 3 is the quinonoid intermediate formed by deprotonation of the external aldimine with l-serine (III in Scheme 1). Further support for the model of Equation 3 came from the direct observation of V in Scheme 1. When H159A SPT was reacted with KDS, a new absorption band appeared at 505 nm, indicating the formation of the quinonoid intermediate (Fig. 5A). The shape and position of the quinonoid intermediate closely matched those of D obtained by global fitting (Fig. 4B). The spectrum of H159A SPT saturated with KDS was not obtained because of the water insolubility of KDS and the spectral deterioration caused by the glycerol used to dissolve KDS (indicated by the increase in the baseline absorption). Using the Kd value of 13 μm (k+4/k−4) for KDS and the concentration of KDS used here (6 μm), the molar absorptivity of the quinonoid species generated from KDS is calculated to be 34,800 m−1 cm−1, comparable with that of D. These results support the idea that D is the deprotonated species of the PLP-KDS external aldimine, i.e. D is equivalent to V in Scheme 1. The absorbance spectra of WT SPT with KDS showed the formation of the external aldimine, but the quinonoid intermediate is only slightly generated (Fig. 5B). The spectrum of the quinonoid intermediate formed by the reaction of H159A SPT with KDS was not stable, and the intensity of the 505-nm absorption gradually decreased with a concomitant increase in the absorption at 332 nm (Fig. 5C). The increase in the 332-nm band was a biphasic process, in which the fast phase corresponded to the decrease in the 505-nm absorption band. These results are interpreted as the decay of the quinonoid intermediate proceeding through a relatively fast protonation to yield the ketimine, followed by a slow hydrolysis to PMP and a ketone. H159A SPT was o