Title: The Role of His-134, -147, and -150 Residues in Subunit Assembly, Cofactor Binding, and Catalysis of Sheep Liver Cytosolic Serine Hydroxymethyltransferase
Abstract: In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed. H134N and H147N SHMTs had K m values for l-serine,l-allo-threonine and β-phenylserine similar to that of wild type enzyme, although the k catvalues were markedly decreased. H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5′-phosphate (PLP) compared with the wild type enzyme. Increasing concentrations of PLP (up to 500 μm) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions. H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding. Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface. H150N SHMT was obtained in a tetrameric form with bound PLP. However, the mutant had very little enzyme activity (<2%). Thek cat/K m values forl-serine, l-allo-threonine and β-phenylserine were 80-, 56-, and 33-fold less compared with wild type enzyme. Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H4-folate. However, it could form an external aldimine with serine and glycine. The wild type and the mutant enzyme had similarK d values for serine and glycine. These results suggest that His-150 may be the base that abstracts the α-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT. In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed. H134N and H147N SHMTs had K m values for l-serine,l-allo-threonine and β-phenylserine similar to that of wild type enzyme, although the k catvalues were markedly decreased. H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5′-phosphate (PLP) compared with the wild type enzyme. Increasing concentrations of PLP (up to 500 μm) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions. H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding. Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface. H150N SHMT was obtained in a tetrameric form with bound PLP. However, the mutant had very little enzyme activity (<2%). Thek cat/K m values forl-serine, l-allo-threonine and β-phenylserine were 80-, 56-, and 33-fold less compared with wild type enzyme. Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H4-folate. However, it could form an external aldimine with serine and glycine. The wild type and the mutant enzyme had similarK d values for serine and glycine. These results suggest that His-150 may be the base that abstracts the α-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT. Serine hydroxymethyltransferase (SHMT) 1The abbreviations used are: SHMT, serine hydroxymethyltransferase; rSHMT, sheep liver cytosolic recombinant SHMT; H4-folate, 5,6,7,8-tetrahydrofolate; PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate. catalyzes the reversible conversion of serine and 5,6,7,8-tetrahydrofolate (H4-folate) to glycine and 5,10-methylene-H4-folate (5,10-CH2-H4-folate) and plays a major role in one-carbon metabolism (1Blakely R.L. Biochem. J. 1955; 61: 315-323Crossref PubMed Scopus (43) Google Scholar). This enzyme is a component of thymidylate cycle, along with thymidylate synthase and dihydrofolate reductase and has been suggested as an alternate target for cancer chemotherapy (2Appaji Rao N. Ramesh K.S. Manohar R. Rao D.N. Vijayalakshmi D. Bhaskaran N. J. Sci. Indust. Res. ( India ). 1987; 46: 248-260Google Scholar, 3Snell K. Natsumeda Y. Eble J.N. Glover J.L. Webber G. Br. J. Cancer. 1988; 57: 87-90Crossref PubMed Scopus (102) Google Scholar, 4Snell K. Bannasch P. Keppler D. Webber G. Liver Carcinoma. Academic Press, Inc., New York1989: 375-387Google Scholar). SHMT contains covalently bound pyridoxal 5′-phosphate (PLP), which forms an internal aldimine with the ε-amino group of Lys-256 in the rabbit and sheep liver cytosolic SHMT (5Martini F. Angelaccio S. Pascarella S. Barra D. Bossa F. Schirch V. J. Biol. Chem. 1987; 262: 5499-5509Abstract Full Text PDF PubMed Google Scholar, 6Usha, R. (1992) The Primary Structure and Active Site Residues of Sheep Liver Serine Hydroxymethyl Transferase. Ph. D. thesis, Indian Institute of Science, Bangalore, IndiaGoogle Scholar). An early step in the catalysis is the formation of an external aldimine, i.e.cofactor-substrate complex, which absorbs between 420–430 nm. This is followed by the formation of a resonance-stabilized carbanion (quinonoid intermediate) with an absorption maximum near 500 nm (7Schirch L. Adv. Enzymol. Relat. Areas Mol. Biol. 1982; 53: 83-112PubMed Google Scholar). The formation of the quinonoid intermediate requires the abstraction of an α-proton from the amino acid substrate. It has been suggested in several PLP enzymes that lysine, which forms an internal aldimine, is the likely candidate for the abstraction of this proton (8Smith D.L. Almo S.C. Toney M.D. Ringe D. Biochemistry. 1989; 28: 8161-8167Crossref PubMed Scopus (103) Google Scholar, 9Toney M.D. Kirsch J.F. Protein Sci. 1992; 1: 109-119Crossref Scopus (58) Google Scholar, 10Bhatia M.B. Futaki S. Ueno H. Manning J.M. Ringe D. Yoshimura T. Soda K. J. Biol. Chem. 1993; 268: 6932-6938Abstract Full Text PDF PubMed Google Scholar, 11Ziak M. Jager J. Malashkevich V.N. Gehring H. Jaussi R. Jansonius J.N. Christen P. Eur. J. Biochem. 1993; 211: 475-484Crossref PubMed Scopus (31) Google Scholar, 12Lu Z. Nagata S. McPhie P. Miles E.W. J. Biol. Chem. 1993; 268: 8727-8734Abstract Full Text PDF PubMed Google Scholar). In contrast to these observations, it has been suggested that inEscherichia coli SHMT, Lys-229 (equivalent to Lys-256 of rabbit and sheep liver cytosolic SHMTs) is not the base that removes the α-proton (13Schirch D. Fratte S.D. Iurescia S. Angelaccio S. Contestabile R. Bossa F. Schirch V. J. Biol. Chem. 1993; 268: 23132-23138Abstract Full Text PDF PubMed Google Scholar). SHMT has been classified by sequence alignments in the same family as aminotransferases for which crystal data are available (14Pascarella S. Schirch V. Bossa F. FEBS Lett. 1993; 331: 145-149Crossref PubMed Scopus (30) Google Scholar, 15Alexander F.W. Sandmeier E. Mehta P.K. Christen P. Eur. J. Biochem. 1994; 219: 953-960Crossref PubMed Scopus (346) Google Scholar, 16Grishin N.V. Phillips M.A. Goldsmith E.J. Protein Sci. 1995; 4: 1291-1304Crossref PubMed Scopus (342) Google Scholar). However, the mechanism of reaction catalyzed by SHMT is different from that of aspartate aminotransferase, since it involves a Cα–Cβ bond cleavage and removal of proton from β-hydroxyl group of serine in the aldol cleavage reaction. In the reverse reaction, with glycine and 5,10-CH2-H4-folate, the 2-S proton (Cα-H) is abstracted to form the quinonoid intermediate. Chemical modification studies with sheep liver cytosolic SHMT suggested that Lys, Arg, His, and Cys residues are essential for catalysis (17Manohar R. Appaji Rao N. Biochem. J. 1984; 224: 703-707Crossref PubMed Scopus (10) Google Scholar). The alignment of SHMT sequences from several sources indicated that a few of the His residues are conserved among prokaryotic and eukaryotic SHMTs (18Usha R. Savithri H.S. Appaji Rao N. Biochim. Biophys. Acta. 1994; 1204: 75-83Crossref PubMed Scopus (26) Google Scholar). As a first step in identifying the His residue(s) essential for enzyme activity, His-147, His-150, which were conserved among all SHMTs, and His-134, which was present only in eukaryotic SHMTs (TableI), were chosen for this study. An inspection of the alignment of the fold type I PLP-dependent enzymes showed that His-147 of sheep liver SHMT corresponds to conserved Trp-140 of aspartate aminotransferase, shown to be involved in PLP binding (19Hayashi H. Inoue Y. Kuramitsu S. Morino Y. Kagamiyama H. Biochem. Biophys. Res. Commun. 1990; 167: 407-412Crossref PubMed Scopus (38) Google Scholar). In other members of the fold type I group, this position is occupied by Phe, Tyr, or His (16Grishin N.V. Phillips M.A. Goldsmith E.J. Protein Sci. 1995; 4: 1291-1304Crossref PubMed Scopus (342) Google Scholar). However, SHMT His-150 is not conserved in other fold type I enzymes except aspartate aminotransferase and glutamate-1-semialdehyde aminotransferase. 2Grishin, N. V., Phillips, M. A., and Goldsmith, E. J. (1995) Protein Sci. 4 (Internet address: gopher://gopher.prosci.uci.edu: 70/00/PSvolumes/v4n7/SUPLEMNT/Grishin.SUP/Grishin.seq). This paper describes the construction, expression, and characterization of H134N, H147N, and H150N mutants. A study of the oligomeric structure and the spectral and catalytic properties of these three mutants suggests that His-134 has a role in subunit interactions, His-147 in the cofactor binding, and His-150 in the proton abstraction step of catalysis.Table IA comparison of the amino acid sequences around His-134, -147, and -150 in sheep liver and other SHMTsSourceSequenceReference134 147 150SHEEP cytE P H A R…G G H L T H G18Usha R. Savithri H.S. Appaji Rao N. Biochim. Biophys. Acta. 1994; 1204: 75-83Crossref PubMed Scopus (26) Google Scholar,23Jagath-Reddy J. Ganesan K. Savithri H.S. Datta A. Appaji Rao N. Eur. J. Biochem. 1995; 230: 533-537Crossref PubMed Scopus (27) Google ScholarRAB cytE P H G R…G G H L T H G5Martini F. Angelaccio S. Pascarella S. Barra D. Bossa F. Schirch V. J. Biol. Chem. 1987; 262: 5499-5509Abstract Full Text PDF PubMed Google Scholar,46Byrne P.C. Sanders P.G. Snell K. Biochem. J. 1992; 286: 117-123Crossref PubMed Scopus (22) Google ScholarRAB mitQ P H D R…G G H L T H G47Martini F. Maras B. Tanci P. Angelaccio S. Pascarella S. Barra D. Bossa F. Schirch V. J. Biol. Chem. 1989; 264: 8509-8519Abstract Full Text PDF PubMed Google ScholarHUM cytE P H G R…G G H L T H G48Garrow T.A. Brenner A.A. Whitehead V.M. Chen X.-N. Duncan R.G. Korenberg J.R. Shane B. J. Biol. Chem. 1993; 268: 11910-11916Abstract Full Text PDF PubMed Google ScholarHUM mitQ P H D R…G G H L T H G48Garrow T.A. Brenner A.A. Whitehead V.M. Chen X.-N. Duncan R.G. Korenberg J.R. Shane B. J. Biol. Chem. 1993; 268: 11910-11916Abstract Full Text PDF PubMed Google ScholarPEA mitK P H D R…G G H L S H G49Turner S.R. Ireland R. Morgan C. Rawsthorne S. J. Biol. Chem. 1992; 267: 13528-13534Abstract Full Text PDF PubMed Google ScholarNCRA cytP V H G R…G G H L S H G50McClung C.R. Davis C.R. Page K.M. Denome S.A. Mol. Cell. Biol. 1992; 12: 1412-1421Crossref PubMed Scopus (16) Google ScholarYEAST cytK P H E R…G G H L S H G51McNeil J.B. McIntosh E.M. Taylor B.V. Zhang F. Tang S. Bognar A.L. J. Biol. Chem. 1994; 269: 9155-9165Abstract Full Text PDF PubMed Google ScholarECOLIE P G D T…G G H L T H G52Plamann M.D. Stauffer L.T. Urbanowski M.L. Stauffer G.V. Nucleic Acids Res. 1983; 11: 2065-2075Crossref PubMed Scopus (57) Google ScholarBRAJAQ P G D T…G G H L T H G53Rossbach S. Hennecke H. Mol. Microbiol. 1991; 5: 39-47Crossref PubMed Scopus (21) Google ScholarSALTYQ P G D T…G G H L T H G54Urbanowsky M.L. Plamann M.D. Stauffer L.T. Stauffer G.V. Gene ( Amst. ). 1984; 27: 47-54Crossref PubMed Scopus (16) Google ScholarHYPMEQ P G D T…G G H L T H G55Atsuro M. Toyokazu Y. Kenji Y. Cheiko Y. Tadashi T. Hiroyuki T. Toshio M. Yoshikazu I. Eur. J. Biochem. 1993; 212: 745-750Crossref PubMed Scopus (22) Google ScholarCAMJEN P G D K…G G H L T H G20Chan V.L. Bingham H.L. Gene ( Amst. ). 1991; 101: 51-58Crossref PubMed Scopus (25) Google ScholarPairwise alignment of sheep liver SHMT with all the other SHMTs was made using the GAP program of the Genetics Computer Group (GCG) package. Multiple alignment was made using the PRETTY program of the GCG package. Open table in a new tab Pairwise alignment of sheep liver SHMT with all the other SHMTs was made using the GAP program of the Genetics Computer Group (GCG) package. Multiple alignment was made using the PRETTY program of the GCG package. [α-32P]dATP (3000 Ci/mmol),l-[3-14C]serine (55 mCi/mmol), restriction endonucleases, SequenaseTM version 2.0 DNA sequencing kit, and DNA-modifying enzymes were obtained from Amersham International. CM-Sephadex and Sephacryl S-200 were obtained from Pharmacia. Glycine,l-serine, d-alanine, NADH, β-phenylserine, 2-mercaptoethanol, folic acid, PLP, and EDTA were obtained from Sigma.l-allo-threonine was purchased from Fluka. H4-Folate was prepared by the method of Hatefi et al. (21Hatefi Y. Talbert P.T. Osborn M.J. Huennekens F.M. Biochem. Prep. 1959; 7: 89-92Google Scholar). All other chemicals were of analytical reagent grade. The mutant oligonucleotides were purchased from Bangalore Genei Private Ltd., Bangalore, India. Centricon filters were obtained from Amicon, Inc. The Altered Sites II in vitro mutagenesis system was purchased from Promega Corp. E. colistrain DH5α (Life Technologies, Inc.) was the recipient for all plasmids used in subcloning. The BL21(DE3) pLysS strain (22Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4834) Google Scholar) was used for bacterial expression of pETSH (23Jagath-Reddy J. Ganesan K. Savithri H.S. Datta A. Appaji Rao N. Eur. J. Biochem. 1995; 230: 533-537Crossref PubMed Scopus (27) Google Scholar) and His mutant constructs. Luria-Bertani medium or terrific broth (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) with 50 μg/ml of ampicillin was used for growing E. coli cells containing the plasmids (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Plasmids were prepared by the alkaline lysis procedure as described by Sambrook et al. (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Restriction endonuclease digestions, Klenow filling, and ligations were carried out according to the manufacturer's instructions. The preparation of competent cells and transformation was carried out by the method of Alexander (25Alexander D.C. Methods Enzymol. 1987; 154: 41-64Crossref PubMed Scopus (33) Google Scholar). From the agarose gel, the DNA fragments were eluted by the low melting agarose gel method (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The sheep liver cytosolic SHMT cDNA clone was isolated and overexpressed in E. coli(23Jagath-Reddy J. Ganesan K. Savithri H.S. Datta A. Appaji Rao N. Eur. J. Biochem. 1995; 230: 533-537Crossref PubMed Scopus (27) Google Scholar). This clone was used for the preparation of site-specific mutants described in this paper. H134N and H147N mutants were constructed using a polymerase chain reaction-based megaprimer method as described earlier (26Jagath-Reddy J. Appaji Rao N. Savithri H.S. Curr. Sci. 1996; 71: 710-712Google Scholar). These mutants were constructed from pUCSH (containing the SHMT cDNA fragment lacking 227 bp at the 5′-end in a pUC 19 vector) as a template. The mutant oligonucleotides, 5′-G GTG GAG CCCAAT GGC CGC A-3′ and 5′-G GAT GGG GGC AAC CTG ACC C 3′ were used for the construction of the H134N and H147N mutants, respectively. The full-length polymerase chain reaction products, obtained upon two rounds of polymerase chain reaction were subcloned into the pUC 19 vector at KpnI and BamHI sites. The clones obtained after the mutagenesis procedure were screened by sequencing the gene at the mutated region. The H150N mutant was generated using the Altered Sites II in vitro mutagenesis system from Promega. This mutant was constructed using a 20-mer mutagenic primer (5′-C CAC CTG ACC AAT GGG TTC A-3′) from the pALSH clone (SHMT cDNA clone lacking 227 bp at the 5′-end in pALTER-1 vector). Initially, the clones were screened by ampicillin selection and later by DNA sequencing according to the mutagenesis kit protocol. pUC 19 and pALTER-1 plasmids containing the mutated SHMT cDNA were purified and digested using the KpnI andPmaCI restriction enzymes flanking all three histidine mutations. The 520-base pair KpnI-PmaCI mutated DNA fragments were gel-purified and swapped at the same sites of pETSH vector (23Jagath-Reddy J. Ganesan K. Savithri H.S. Datta A. Appaji Rao N. Eur. J. Biochem. 1995; 230: 533-537Crossref PubMed Scopus (27) Google Scholar). The clones obtained were screened by sequencing. The entire 520-base pair KpnI-PmaCI DNA fragments were sequenced using SequenaseTM version 2.0 DNA sequencing kit in all three mutants to rule out the presence of other nonspecific mutations. pETSH, H134N, H147N, and H150N mutant enzymes were purified as described by Jagath et al. by subjecting BL21 (DE3) pLys extracts to ammonium sulfate fractionation, CM-Sephadex, Sephacryl S-200 column chromatography (27Jagath J.R. Sharma B. Bhaskar B. Datta A. Appaji Rao N. Savithri H.S. Eur. J. Biochem. 1997; 247: 372-379Crossref PubMed Scopus (30) Google Scholar). Sephacryl S-200 fractions containing SHMT were pooled and precipitated with 65% ammonium sulfate. The pellet was resuspended in buffer A (50 mmpotassium phosphate buffer, pH 7.4, containing 1 mm2-mercaptoethanol and 1 mm EDTA) and dialyzed against 1 liter of the same buffer (with two changes) for 24 h. This enzyme preparation was used in these studies. One ml of purified rSHMT (10A 280/ml) enzyme was passed through a Centricon filter by rinsing with 10 ml of double distilled water. After Centricon filtration, the absorbance was measured at 280 nm, and then the sample was lyophilized and weighed. The concentration at 1A 280 was found to be 1.2 mg with a molar extinction coefficient of 176,333 m−1cm−1. A similar value was obtained when the protein concentration was estimated by the method of Gill and von Hippel (28Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5060) Google Scholar). This value was used for the estimation of all three mutant enzymes, since the absorbance at 280 nm did not change compared with wild type enzyme. The SHMT-catalyzed aldol cleavage of serine with H4-folate to form glycine and 5,10-CH2-H4 folate was monitored usingl-[3-14C]serine and H4-folate as substrates in the absence and presence of 500 μm PLP (29Taylor R.T. Weissbach H. Anal. Biochem. 1965; 13: 80-84Crossref Scopus (168) Google Scholar,30Manohar R. Ramesh K.S. Appaji Rao N. J. Biosci. 1982; 4: 31-50Crossref Scopus (30) Google Scholar). The SHMT-catalyzed aldol cleavage ofl-allo-threonine to glycine and acetaldehyde was monitored at 340 nm by the NADH-dependent reduction of acetaldehyde to ethanol and NAD+ in the presence of an excess amount of alcohol dehydrogenase as described earlier (31Malkin L.I. Greenberg D.M. Biochim. Biophys. Acta. 1964; 85: 117-131PubMed Google Scholar) with the following modifications. One ml of buffer A contained SHMT (1–70 μg), alcohol dehydrogenase (100 μg), NADH (250 μm), and 0–10 mml-allo-threonine. The reference cuvette contained all the above components exceptl-allo-threonine. The reaction was monitored in the absence and presence of PLP (500 μm) at 37 °C for 10 min. The NADH consumed in the reaction was calculated using a molar extinction coefficient of 6220 m−1cm−1 (32Ciotti M.M. Kaplan N.O. Methods Enzymol. 1957; 3: 890-899Crossref Scopus (85) Google Scholar). The rate of cleavage of β-phenylserine to benzaldehyde was monitored at 279 nm for 5 min at 37 °C as described earlier (33Ulevitch R.J. Kallen R.G. Biochemistry. 1977; 16: 5342-5349Crossref PubMed Scopus (78) Google Scholar). The amount of the enzyme (rSHMT and mutant enzymes) used in the reaction was in the range of 2.5–300 μg. The concentration of benzaldehyde formed in the reaction was calculated using a molar extinction coefficient of 1400 m−1cm−1. The rates of transamination of d-alanine to pyruvate and pyridoxamine 5′-phosphate (PMP) were determined by measuring the rate of increase in absorbance at 325 nm (34Schirch L. Jenkins W.T. J. Biol. Chem. 1964; 239: 3797-3800Abstract Full Text PDF PubMed Google Scholar). The reaction was carried out in buffer A at 37 °C for 5 min. The amount of PMP formed in the reaction was calculated using a molar extinction coefficient of 8300 m−1 cm−1(35Peterson E.A. Sober H.A. J. Am. Chem. Soc. 1954; 76: 169-183Crossref Scopus (312) Google Scholar). The concentration of the enzyme (rSHMT and H150N SHMT) used in the reaction was in the range of 0.35–1.5 mg. CD measurements were made in Jasco-J-500 A automated recording spectropolarimeter. All CD spectra were recorded at 22 ± 2 °C in buffer A using the same buffer as blank. A protein concentration corresponding to 0.12 mg/ml was used for far-UV CD studies. Visible CD spectra were recorded in a Jasco-J-20 C automated recording spectropolarimeter at a protein concentration of 1.2 mg/ml in buffer A. To analyze the oligomeric structures of the His mutant enzymes, a Superose-12 HR 10/30 analytical gel filtration column attached to Pharmacia FPLC system was used. The column was calibrated with standard proteins such as apoferritin (440 kDa), sheep cytosolic SHMT (213 kDa), yeast alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). The buffer used for this study was buffer A containing 0.1 m KCl and 0.05% sodium azide. The dissociation constants of the enzyme-substrate complexes with rSHMT and H150N SHMT were determined by a slight modification of the earlier procedure (36Schirch L. Diller A. J. Biol. Chem. 1971; 246: 3961-3966Abstract Full Text PDF PubMed Google Scholar). The substrate-induced quenching of enzyme bound PLP fluorescence was monitored using a Shimadzu RF-5000 spectrofluorimeter. The enzyme (1 mg/ml) sample was incubated with increasing concentrations ofl-serine (0.1–50 mm) or glycine (0.5–100 mm) at 25 ± 2 °C, and the fluorescence was monitored at 450–550 nm after excitation at 425 nm. TheK d values were obtained from double reciprocal plots of the change in fluorescence units at 495 nm as a function of the ligand concentration. It was ensured that inner filter quenching was minimal and did not interfere with the measurements. [2-3H]Glycine was purified on Dowex-50W-12 column packed in a 1-ml syringe as described (37Chen M.S. Schirch L. J. Biol. Chem. 1973; 248: 3631-3635Abstract Full Text PDF PubMed Google Scholar). The rSHMT or H150N enzyme (60 μg) in HEPES buffer pH 7.4 was incubated with 30 mm [2-3H]glycine (2.2 × 105 cpm) for 10 min at 37 °C. After the incubation, H4-folate (0–100 μm) was added, and the reaction continued for an additional 1 min at 37 °C. The reaction was stopped by the addition of 10% trichloroacetic acid, and the denatured protein was removed by centrifugation. The supernatant was loaded onto a Dowex 50W-12 column that was previously equilibrated with 10 mm HCl. The column was washed with 5 ml of 10 mm HCl, the eluant was collected (0.5-ml fractions), and the radioactivity was measured. Apoenzyme of rSHMT was prepared as described earlier (13Schirch D. Fratte S.D. Iurescia S. Angelaccio S. Contestabile R. Bossa F. Schirch V. J. Biol. Chem. 1993; 268: 23132-23138Abstract Full Text PDF PubMed Google Scholar) with minor modifications. d-Alanine (200 mm) was added to the holoenzyme in 50 mmpotassium phosphate buffer, pH 7.4, containing 10 mm2-mercaptoethanol, 1 mm EDTA and 200 mmammonium sulfate and incubated at 37 °C for 4 h. The pyruvate and PMP formed during the reaction were removed by passing the sample through a 30-kDa centricon filter. The conserved His-134, -147, and -150 residues were mutated to Asn by site-directed mutagenesis as described under "Experimental Procedures." The expression of the mutant constructs was as good, since the wild type clone (pETSH) and the expressed proteins were present predominantly (>90%) in the soluble fraction. The enzyme present in the soluble fraction was purified by a procedure identical to that used for the wild type enzyme, and yields of the enzymes were in the range of 40–50 mg/liter. The purified mutant proteins were homogeneous as indicated by a single band on native PAGE and SDS-polyacrylamide gel electrophoresis. The purified rSHMT (wild type), H134N, H147N, and H150N SHMTs were assayed using 0.6, 15, 30, and 30 μg of the enzyme, respectively. The H150N SHMT had the lowest specific activity of 0.06 units/mg, while H134N and H147N SHMTs had 0.18 and 0.09 units/mg, respectively, compared with a value of 4.8 units/mg for rSHMT. The far-UV CD spectra of all the mutant enzymes were essentially similar to the wild type enzyme, suggesting that there were no alterations in the secondary structure upon mutation of the specified His to Asn residues. The presence of characteristic spectral intermediates in the catalytic process of SHMT has provided a convenient handle to examine the specific functions of identified amino acid residues in the structure and function of the enzyme. It can be seen from Fig.1 a that H150N has slightly reduced absorbance at 425 nm compared with rSHMT, while H134N and H147N SHMTs had much less absorbance when an equal concentration of protein (1.2 mg) was used for recording the spectrum. H134N and H147N SHMTs had very little CD in the visible region (350–500 nm), while H150N SHMT gave a visible CD spectrum characteristic of the presence of an internal aldimine at the active site. H134N, H147N, and H150N SHMTs had approximately 6.3, 3, and 64% of the visible CD (at 425 nm) that rSHMT had, respectively (data not shown). It can be seen from Fig.1 b that the quinonoid intermediate (495 nm) was observed when glycine and H4-folate were added to H134N and H147N mutant enzymes. However, this intermediate was not seen with H150N SHMT. Even increasing the concentration of H150N SHMT from 1 to 5 mg did not result in the formation of this intermediate. The oligomeric status of the enzymes immediately after the Sephacryl-S-200 column chromatography step of purification was examined by using a calibrated Superose-12 HR 10/30 analytical gel filtration column. The rSHMT, H147N, and H150N SHMTs eluted as single symmetrical peak corresponding to a molecular mass of ∼220 kDa, indicating that they are in the tetrameric form. However, the H134N SHMT eluted as a single peak corresponding to the mass of the dimer (∼100 kDa) (Fig. 2 a). When the column was equilibrated with PLP (150 μm), the elution profiles were identical. Dialysis of the H147N mutant enzyme against the buffer not containing the PLP resulted in the formation of apoenzyme that was predominantly in the dimeric form; in contrast, the rSHMT remained as a tetramer with bound PLP under similar conditions (Fig. 2 b). Removal of PLP from rSHMT by transamination ofd-alanine followed by dialysis for 24 h resulted in the partial dissociation of the tetramer to a dimer (Fig.2 b). A unique feature of SHMT is its ability to catalyze a variety of H4-folate-dependent and -independent reactions (7Schirch L. Adv. Enzymol. Relat. Areas Mol. Biol. 1982; 53: 83-112PubMed Google Scholar). To assess the role of the mutated His residues in reaction specificity, some of these reactions were examined. Table II summarizes the K m,k cat andk cat/K m values determined for H134N, H147N, and H150N SHMTs using l-serine andl-allo-threonine as substrates in the absence and presence of added PLP (0.5 mm). K mvalues of serine and H4-folate (1.0 ± 0.2 mm) for the mutant enzymes were similar to that of wild type enzyme, whereas the k cat values decreased significantly for all of the three mutant enzymes. A comparison of thek cat/K m values indicated that the H134N, H147N, and H150N SHMTs are 36-, 70-, and 80-fold less efficient compared with the wild type enzyme in the H4-folate-dependent physiological reaction in the absence of added PLP, whereas in the presence of 500 μm PLP, the activities were 6.85-, 8.64-, and 64-fold less efficient. The K m values forl-allo-threonine were similar in the absence and presence of added PLP for all of the mutant enzymes and rSHMT (TableII). However, the k cat values were markedly decreased. The k cat/K m values for l-allo-threonine with the H134N, H147N, and H150N SHMTs were 21-, 32-, and 60 ± 4-fold less compared with wild type enzyme in the absence and presence of added PLP. The difference in the k cat values of the mutants (H134N, H147N) in the H4-folate-dependent and -independent reactions in the presence of excess PLP could be due to the effect of a large excess PLP or errors in the estimation of the activity due to the interference of PLP absorbance at 340 nm, the wavelength at which alcohol dehydrogenase activity was estimated. The mutant enzymes catalyzed the β-phenylserine cl