Essential Roles of Zinc Ligation and Enzyme Dimerization for Catalysis in the Aminoacylase-1/M20 Family
2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês
10.1074/jbc.m304233200
ISSN1083-351X
AutoresHolger A. Lindner, V.V. Lunin, Alain Alary, Regina Hecker, Mirosław Cygler, Robert Ménard,
Tópico(s)Enzyme Structure and Function
ResumoMembers of the aminoacylase-1 (Acy1)/M20 family of aminoacylases and exopeptidases exist as either monomers or homodimers. They contain a zinc-binding domain and a second domain mediating dimerization in the latter case. The roles that both domains play in catalysis have been investigated for human Acy1 (hAcy1) by x-ray crystallography and by site-directed mutagenesis. Structure comparison of the dinuclear zinc center in a mutant of hAcy1 reported here with dizinc centers in related enzymes points to a difference in zinc ligation in the Acy1/M20 family. Mutational analysis supports catalytic roles of zinc ions, a vicinal glutamate, and a histidine from the dimerization domain. By complementing different active site mutants of hAcy1, we show that catalysis occurs at the dimer interface. Reinterpretation of the structure of a monomeric homolog, peptidase V, reveals that a domain insertion mimics dimerization. We conclude that monomeric and dimeric Acy1/M20 family members share a unique active site architecture involving both enzyme domains. The study may provide means to improve homologous carboxypeptidase G2 toward application in antibody-directed enzyme prodrug therapy. Members of the aminoacylase-1 (Acy1)/M20 family of aminoacylases and exopeptidases exist as either monomers or homodimers. They contain a zinc-binding domain and a second domain mediating dimerization in the latter case. The roles that both domains play in catalysis have been investigated for human Acy1 (hAcy1) by x-ray crystallography and by site-directed mutagenesis. Structure comparison of the dinuclear zinc center in a mutant of hAcy1 reported here with dizinc centers in related enzymes points to a difference in zinc ligation in the Acy1/M20 family. Mutational analysis supports catalytic roles of zinc ions, a vicinal glutamate, and a histidine from the dimerization domain. By complementing different active site mutants of hAcy1, we show that catalysis occurs at the dimer interface. Reinterpretation of the structure of a monomeric homolog, peptidase V, reveals that a domain insertion mimics dimerization. We conclude that monomeric and dimeric Acy1/M20 family members share a unique active site architecture involving both enzyme domains. The study may provide means to improve homologous carboxypeptidase G2 toward application in antibody-directed enzyme prodrug therapy. Zinc peptidases play roles in metabolic and signaling pathways throughout all kingdoms of life. A growing number of these enzymes have been found to contain two zinc ions at their active sites. Some are regarded as potential pharmaceutical targets (1Holz R.C. Bzymek K.P. Swierczek S.I. Curr. Opin. Chem. Biol. 2003; 7: 197-206Crossref PubMed Scopus (56) Google Scholar). Recently, Wouters and Husain (2Wouters M.A. Husain A. J. Mol. Biol. 2001; 314: 1191-1207Crossref PubMed Scopus (30) Google Scholar) pointed out that members of the MH and MF clans 1This report follows the MEROPS classification of peptidases (available on the World Wide Web at merops.sanger.ac.uk) (3Rawlings N.D. O'Brien E. Barrett A.J. Nucleic Acids Res. 2002; 30: 343-346Crossref PubMed Scopus (177) Google Scholar). This system groups peptidases with significant sequence similarity into a family and assigns families of common evolutionary origin to a clan. of dizinc peptidases, together with the MC clan of monozinc peptidases display three different catalytic zinc centers that have evolved in a similar structural scaffold, which is exemplified by carboxypeptidase A of clan MC. Although they all appear to employ the same general base-like catalytic mechanism, neither all catalytic residues nor the substrate-binding sites are conserved among the three clans. A glutamate residue representing a putative catalytic base, for instance, resides in different regions of the polypeptide chain in the MH and MC clans. In the MF clan enzyme leucine aminopeptidase, a bicarbonate ion replaces the glutamate residue. Moreover, whereas the monozinc center in the MC clan is structurally equivalent to one of the two zincbinding sites in the dinuclear zinc center in the MH clan, it does not share any homology with the dinuclear zinc center in the MF clan anymore. As a consequence, members of families from each clan must be examined individually in order to gain a detailed understanding of their catalytic machineries. Aminoacylase-1 (Acy1 2The abbreviations used are: Acy1, aminoacylase 1; CPG2, Pseudomonas sp. carboxypeptidase G2; PepT, amino tripeptidase T; PepV, peptidase V; ADEPT, antibody-directed prodrug therapy; AAP, Aeromonas proteolytica aminopeptidase; SGAP, Streptomyces griseus aminopeptidase; hAcy1, human Acy1; r.m.s., root mean square.; EC 3.5.1.14) was discovered in 1881 by virtue of its ability to hydrolyze hippuric acid in crude kidney homogenates (4Schmiedeberg O. Naunym-Schmiedberg's Arch. Exp. Pathol. Pharmakol. 1881; 14: 379-392Crossref Scopus (24) Google Scholar) and is now classified in the M20 family of clan MH, also referred to as the Acy1 family (5Biagini A. Puigserver A. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2001; 128: 469-481Crossref PubMed Scopus (32) Google Scholar). Acy1 plays a general role in the cytosolic breakdown of N α-acetylated amino acids (6Gade W. Brown J.L. Biochim. Biophys. Acta. 1981; 662: 86-93Crossref PubMed Scopus (49) Google Scholar) generated during protein degradation (7Jones W.M. Scaloni A. Bossa F. Popowicz A.M. Schneewind O. Manning J.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2194-2198Crossref PubMed Scopus (46) Google Scholar). Other functional aminoacylase enzymes from the Acy1/M20 family (Table I) are implicated in the bacterial biosynthesis pathways of arginine (N-acetylornithine deacetylase), lysine, and the cell wall (succinyldiaminopimelate desuccinylase) (8Boyen A. Charlier D. Charlier J. Sakanyan V. Mett I. Glansdorff N. Gene (Amst.). 1992; 116: 1-6Crossref PubMed Scopus (38) Google Scholar). Several enzymes of the Acy1/M20 family are known as exopeptidases (Table I). They include CPG2 (9Levy C.C. Goldman P. J. Biol. Chem. 1967; 242: 2933-2938Abstract Full Text PDF PubMed Google Scholar, 10Goldman P. Levy C.C. Proc. Natl. Acad. Sci. U. S. A. 1967; 58: 1299-1306Crossref PubMed Scopus (45) Google Scholar, 11Sherwood R.F. Melton R.G. Alwan S.M. Hughes P. Eur. J. Biochem. 1985; 148: 447-453Crossref PubMed Scopus (194) Google Scholar), Saccharomyces cerevisiae carboxypeptidase Y (12Spormann D.O. Heim J. Wolf D.H. J. Biol. Chem. 1992; 267: 8021-8029Abstract Full Text PDF PubMed Google Scholar), PepT and the dipeptidase enzyme PepV from various bacterial sources (13Vongerichten K.F. Klein J.R. Matern H. Plapp R. Microbiology. 1994; 140: 2591-2600Crossref PubMed Scopus (47) Google Scholar, 14Barrett A.J. Rawlings N.D. Woessner J.F. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 1412-1416Google Scholar, 15Hellendoorn M.A. Franke-Fayard B.M. Mierau I. Venema G. Kok J. J. Bacteriol. 1997; 179: 3410-3415Crossref PubMed Google Scholar), Escherichia coli X-His dipeptidase (16Schroeder U. Henrich B. Fink J. Plapp R. FEMS Microbiol. Lett. 1994; 123: 153-159Crossref PubMed Scopus (23) Google Scholar), and human nonspecific dipeptidase and a brain-specific carnosinase that possibly plays a role in aging and neurodegenerative or psychiatric diseases (17Teufel M. Saudek V. Ledig J.P. Bernhardt A. Boularand S. Carreau A. Cairns N.J. Carter C. Cowley D.J. Duverger D. Ganzhorn A.J. Guenet C. Heintzelmann B. Laucher V. Sauvage C. Smirnova T. J. Biol. Chem. 2003; 278: 6521-6531Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). The enzymatic function of a related drought-induced polypeptide-1 from wild watermelon (Citrullus lanatus) remains unknown (18Kawasaki S. Miyake C. Kohchi T. Fujii S. Uchida M. Yokota A. Plant Cell Physiol. 2000; 41: 864-873Crossref PubMed Scopus (112) Google Scholar).Table IRepresentative enzymes of the M20 and M28 familiesEnzymeMEROPS identifieraThe first part of the MEROPS identifier indicates the family assignment.N-Acylamino acid amidohydrolasesHuman aminoacylase-1 (l-acylamino acid amidohydrolase)M20.973Bacterial N-acetylornithine deacetylaseM20 nonpeptidase homologueBacterial succinyldiaminopimelate desuccinylaseM20 nonpeptidase homologueCarboxypeptidasesPseudomonas sp. carboxypeptidase G2M20.001S. cerevisiae carboxypeptidase YM20.002Human glutamate carboxypeptidase IIM28.010Amino tripeptidases and dipeptidasesBacterial amino tripeptidase TM20.003Bacterial peptidase VM20.004E. coli X-His dipeptidaseM20.007Human nonspecific dipeptidaseM20.005Human carnosinaseM20.006A. proteolytica aminopeptidaseM28.002S. griseus aminopeptidaseM28.003Specificity unknownCitrullus lanatus drought-induced polypeptideM20 nonpeptidase homologuea The first part of the MEROPS identifier indicates the family assignment. Open table in a new tab Enzymes of the Acy1/M20 family have shown potential for different applications. In biocatalysis, the high stereoselectivity of Acy1 allows the preparation of l-amino acids from racemic mixtures of N-acyl-l-amino acids (19Bommarius A.S. Drauz K. Waldmann H. Enzyme Catalysis in Organic Synthesis. Wiley-VCH, Weinheim2002: 741-749Crossref Scopus (3) Google Scholar). Succinyldiaminopimelate desuccinylase is considered as a potential anti-bacterial target (1Holz R.C. Bzymek K.P. Swierczek S.I. Curr. Opin. Chem. Biol. 2003; 7: 197-206Crossref PubMed Scopus (56) Google Scholar), and CPG2 is considered a therapeutic agent in ADEPT for cancer treatment (18Kawasaki S. Miyake C. Kohchi T. Fujii S. Uchida M. Yokota A. Plant Cell Physiol. 2000; 41: 864-873Crossref PubMed Scopus (112) Google Scholar). However, compared with the well characterized active sites of AAP (21Chevrier B. Schalk C. D'Orchymont H. Rondeau J.M. Moras D. Tarnus C. Structure. 1994; 2: 283-291Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar) and SGAP (22Gilboa R. Spungin-Bialik A. Wohlfahrt G. Schomburg D. Blumberg S. Shoham G. Proteins. 2001; 44: 490-504Crossref PubMed Scopus (50) Google Scholar) from the M28 family of clan MH (Table I), relatively little is known about the Acy1/M20 family. The crystal structures of two Acy1 homologs, CPG2 (23Rowsell S. Pauptit R.A. Tucker A.D. Melton R.G. Blow D.M. Brick P. Structure. 1997; 5: 337-347Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) and PepT from Salmonella typhimurium (24Hakansson K. Miller C.G. Eur. J. Biochem. 2002; 269: 443-450Crossref PubMed Scopus (45) Google Scholar), are each folded into a metal-binding domain and a smaller dimerization domain, which is inserted in the middle of the sequence of the metal-binding domain. In the structures of both enzymes, the two domains display an open conformation. Porcine Acy1 was shown by limited proteolysis to have a closely similar domain structure (25Palm G.J. Rohm K.H. J. Protein Chem. 1995; 14: 233-240Crossref PubMed Scopus (22) Google Scholar, 26Lindner H. Berens W. Kraus I. Röhm K.H. Biol. Chem. 2000; 381: 1055-1061Crossref PubMed Scopus (3) Google Scholar). The metalbinding domains in CPG2 and PepT exhibit high structural similarity to the two single-domain proteins AAP and SGAP and are thought to be responsible for catalysis. Besides CPG2 and PepT, numerous other members of the Acy1/M20 family, including Acy1, appear to exist as homodimers (8Boyen A. Charlier D. Charlier J. Sakanyan V. Mett I. Glansdorff N. Gene (Amst.). 1992; 116: 1-6Crossref PubMed Scopus (38) Google Scholar, 17Teufel M. Saudek V. Ledig J.P. Bernhardt A. Boularand S. Carreau A. Cairns N.J. Carter C. Cowley D.J. Duverger D. Ganzhorn A.J. Guenet C. Heintzelmann B. Laucher V. Sauvage C. Smirnova T. J. Biol. Chem. 2003; 278: 6521-6531Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 26Lindner H. Berens W. Kraus I. Röhm K.H. Biol. Chem. 2000; 381: 1055-1061Crossref PubMed Scopus (3) Google Scholar, 27Kördel W. Schneider F. Z. Naturforsch. C. 1977; 32: 342-344Crossref PubMed Scopus (53) Google Scholar, 28Bosman B.W. Tan P.S.T. Konings W.N. Appl. Environ. Microbiol. 1990; 56: 1839-1843Crossref PubMed Google Scholar). It is not clear whether the dimerization domain in these enzymes is involved in catalysis. Recently, the crystal structure of PepV from Lactobacillus delbrueckii revealed that the enzyme exists as a monomer (29Jozic D. Bourenkow G. Bartunik H. Scholze H. Dive V. Henrich B. Huber R. Bode W. Maskos K. Structure. 2002; 10: 1097-1106Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). As in the dimer forming enzymes of the Acy1/M20 family, the additional domain in PepV is inserted in the zinc-binding domain and is referred to as the lid domain. The structure of PepV was determined in a complex with an inhibitor, AspΨ[PO2CH2]AlaOH, which mimics the transition state of a dipeptide substrate, bound in a hydrophobic cavity between the zinc-binding and the lid domain (29Jozic D. Bourenkow G. Bartunik H. Scholze H. Dive V. Henrich B. Huber R. Bode W. Maskos K. Structure. 2002; 10: 1097-1106Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Its interactions with the enzyme pinpoint the active site residues. In addition to residues from the zinc-binding domain, residues from the lid domain also appear to be involved in substrate binding and catalysis. Here, we report the crystal structure of the metal-binding domain of a mutant of hAcy1. A spatial comparison of MH clan enzymes showed high structural conservation of the dinuclear zinc center but also revealed a difference in zinc ligation, which correlates with differences in substrate specificity among the known enzymes of the Acy1/M20 family. Mutational studies of hAcy1 suggest that both zinc sites and a conserved glutamate in their immediate vicinity are essential for catalysis. We further recognized that the lid domain in the related but monomeric enzyme PepV mimics the structure of a dimer and thereby inserts a catalytic histidine into the active site. Mutational analysis of the corresponding histidine in the small domain of hAcy1 supports the role of this residue in catalysis in the dimeric enzyme. An enzyme complementation assay provides evidence that the histidine is acting in trans and that catalysis occurs at the dimer interface. Site-directed Mutagenesis—Site-directed mutagenesis was performed on the baculovirus transfer vector pVL1393-hAcI (30Pittelkow S. Lindner H. Röhm K.H. Protein Expression Purif. 1998; 12: 269-276Crossref PubMed Scopus (24) Google Scholar) using the QuikChange™ XL site-directed mutagenesis kit (Stratagene). Forward versions of the mutagenic primers are listed in Table II. The sequences of the mutant hAcy1 genes in the resulting transfer vectors were confirmed by DNA sequencing.Table IIForward versions of mutagenic primersMutantPredicted role in hAcy1Oligonucleotide sequenceaAltered nucleotides are underlined.E148AZinc binding5′-GCCTGATGAGGCGGTTGGGGGTCACC-3′H373AZinc binding5′-CCTGTGCTGCTGGCCGACCACGATGAACGGC-3′H80AZinc binding5′-CTCAACTCCGCCACGGATGTGGTGCCTGTCTTCAAG-3′E175AZinc binding5′-GGCTTTGCCCTGGATGCGGGCATAGCCAATCC-3′D113AZinc binding5′-GGGGTGCCCAGGCCATGAAGTGCGTCAGC-3′E147AGeneral base5′-CCTTTGTGCCTGATGCGGAGGTTGGGGG-3′E147Q5′-CCTTTGTGCCTGATCAGGAGGTTGGGGG-3′E147D5′-CCTTTGTGCCTGATGACGAGGTTGGGGG-3′H206NCatalytic5′-GGGAGGCCAGGCAATGCCTCACGCTTC-3′T347G5′-GCCTGCTGCCGGTGACAACCGCTATATCCG-3′a Altered nucleotides are underlined. Open table in a new tab Enzyme Expression and Purification—Wild-type hAcy1 and its variants were expressed in a baculovirus expression vector system and purified as described (30Pittelkow S. Lindner H. Röhm K.H. Protein Expression Purif. 1998; 12: 269-276Crossref PubMed Scopus (24) Google Scholar). In brief, 300-ml cultures of infected Sf21 cells were harvested 72 h after infection. A two-step purification protocol was applied in all cases. The enzyme preparations were stored at –80 °C. Before use, protein samples were transferred into the appropriate buffer using a PD-10 column (Amersham Biosciences). Protein was concentrated using Centricon-10 concentrators (Amicon) following the instructions of the supplier. Protein concentrations were determined by the Bradford assay (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) with bovine serum albumin as the standard. Protein Crystallization, Data Collection, and Refinement—Crystals were obtained by the hanging drop vapor diffusion method, equilibrating drops containing 1.5 μl of protein (5–8 mg/ml) in 20 mm Tris-HCl buffer, pH 7.5, and 2.0 μl of reservoir solution (20% (w/v) polyethylene glycol 8000, 0.2 m (NH4)2SO4, 2 mm CoCl2, and 10 mm norleucine) suspended over 1.0 ml of reservoir solution. The crystals belong to the space group P212121 with unit cell dimensions a = 53.53 Å, b = 67.23 Å, c = 146.48 Å and two molecules forming a dimer in the asymmetric unit. Dissolved protein crystals were analyzed by SDS-PAGE, showing protein bands at 25 and 12 kDa (not shown), whereas the full form of the protein is 46 kDa. The remaining 9-kDa fragment was not detectable. Prior to data collection, the crystals were soaked in a cryo-protecting solution of 25% (w/v) polyethylene glycol 8000, 0.2 m (NH4)2SO4, 22.5% (v/v) glycerol and were flash cooled in a cold stream of N2 gas to 100 K. Diffraction data sets for all crystals were collected using a Quantum-4 CCD detector at the X8C beamline, NSLS, Brookhaven National Laboratory (Table III). Data processing and scaling for all data sets were performed with the program HKL2000 (32Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar).Table IIIX-ray diffraction data collection statisticsData setHg-PeakNativeWavelength1.00570.979700Resolution range (Å)50-1.550-1.4Last shell (Å)1.55-1.501.45-1.40R sym0.0740.056Last shell0.3440.503Completeness95.898.4Last shell81.985.9No. of reflections529,534608,259Unique reflections158,345103,337 Open table in a new tab The protein structure was determined by SIRAS phasing using a native data set and the anomalous peak wavelength from a Hg derivative, obtained by overnight soaking in reservoir solution, containing 2 mm thimerosal. Four Hg sites in the asymmetric unit were found using the program SOLVE (33Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). The phases calculated to 1.5 Å with these sites gave an overall figure of merit of 0.25. Electron density modification was applied with the program RESOLVE (34Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 965-972Crossref PubMed Scopus (1636) Google Scholar) with the solvent content set to 0.4. The figure of merit after density modification was 0.51 at 1.5 Å. Approximately 95% of the protein main chain was built automatically with the ARP/WARP package (35Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar) in warpNtrace mode with input experimental phases after RESOLVE. Side chains were inserted using the docking routine from the ARP/WARP package. The model was then adjusted manually using the program O (36Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and refined against a native data set using the program REFMAC version 5.1.08 (37Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) in anisotropic mode with no σ cut-off. During refinement, 1% of the reflections were set aside for the calculation of Rfree. Water molecules were initially added automatically with CNS (38Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) and subsequently by visual inspection of difference maps. The final model has been refined to a R-factor of 0.133 and Rfree of 0.172 at 1.4 Å (Table IV). The model was further analyzed with the program PROCHECK (39Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), which showed that 88.9% of all residues are in the most favored regions, 10.6% are in additionally allowed regions, two residues are in generally allowed regions, and none in a disallowed region of the Ramachandran plot. All stereochemistry parameters checked by PROCHECK are "inside" or "better" compared with the standard values and deviations.Table IVRefinement and overall crystal structure statisticsUsed data setNativeResolution range (Å)50.0-1.4R-factor (R free) (%)13.3 (17.2)No. of non-hydrogen protein atoms4443No. of water molecules665Average B-factor for chain A (B) (Å2)Main chain atoms11.4 (12.8)Side chain atoms14.3 (15.8)Water molecules25.5Metal ions10.7Substrate molecules16.1r.m.s. deviation bond length (Å)0.023r.m.s. deviation bond angle (degrees)1.890Ramachandran plotResidues in most favorable regions (%)89.5Residues in additional regions (%)10.1Residues in disallowed regions (%)0.0 Open table in a new tab Assays of Enzyme Activity and Stability—Acy1 activity was determined at pH 7.4 by a discontinuous colorimetric assay as described before (30Pittelkow S. Lindner H. Röhm K.H. Protein Expression Purif. 1998; 12: 269-276Crossref PubMed Scopus (24) Google Scholar). Kinetic data were evaluated by nonlinear regression analysis using both the Michaelis-Menten equation (v = V max × [S]/(Km + [S])) and the Hill equation (v = V max × [S] n /([S]0.5n + [S] n)), where [S]0.5 represents the substrate concentration at half-saturation, and the Hill coefficient, n, is a quantitative measure of cooperativity. The catalytic constant, k cat, was calculated using the equation V max = k cat × [E], where [E] represents total enzyme concentration. Kinetic parameters were taken from the equation that generated the best fit to experimental data. As a measure of stability, the "melting temperature," Tm , and the denaturation energy, ΔG(H2O), of each enzyme variant were determined as described previously (26Lindner H. Berens W. Kraus I. Röhm K.H. Biol. Chem. 2000; 381: 1055-1061Crossref PubMed Scopus (3) Google Scholar). Tm values were acquired by monitoring enzymatic inactivation during the slow heating of samples. Aliquots were withdrawn at regular intervals and subjected to enzyme activity determinations. ΔG(H2O) values were estimated by fluorescence equilibrium denaturation on an SPEX Fluorolog-2 spectrofluorometer, using guanidine hydrochloride as the denaturant. Structure-based Alignment and Structure Comparison—Structure-based sequence alignments were derived manually for the zinc-binding domain in the T347G mutant of hAcy1, 3The atomic coordinates for the crystal structure of this protein are available in the Protein Data Bank (http://www.rcsb.org/pdb) under PDB number 1Q7L (40Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27935) Google Scholar). and representative structures of peptidases from the MH clan: CPG2, PepT, PepV, AAP, and SGAP. 4The atomic coordinates for the crystal structures of CPG2, PepT, PepV, AAP, and SGAP are available in the Protein Data Bank (http://www.rcsb.org/pdb) under PDB numbers 1CG2, 1FNO, 1LFW, 1AMP, and 1QQ9, respectively (40Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27935) Google Scholar). Models of the compared proteins were fitted, and r.m.s. calculations for C-α atoms were carried out using the Swiss-PDBviewer (41Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9641) Google Scholar). For some secondary structure elements, additional local fitting was performed manually. Figs. 1, 2, and 3B were made with Raster3D (available on the World Wide Web at www.bmsc.washington.edu/raster3d) (42Merritt A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3878) Google Scholar).Fig. 2Stereo close-up view of the superposition of the zinc centers in the T347G mutant of hAcy1 in complex with glycine, Pseudomonas sp. CPG2 and S. typhimurium PepT (both without ligand), and L. delbrueckii PepV, AAP, and SGAP in complex with AspΨ[PO2CH2]AlaOH, l-leucinephosphonic acid and methionine, respectively. The structures are colored in red, green, brown, cyan, blue, and purple for hAcy1, CPG2, PepT, PepV, AAP, and SGAP, respectively. Only amino acid side chains are shown. The numbering is given for hAcy1. During the superposition, zinc 2 was taken as the first and the zinc 1 as the second fixed point, followed by alignment with the C-γ atom of Asp113.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Structures of the small domains of enzymes from the Acy1/M20 family. A, topology diagram for the lid domain in L. delbrueckii PepV and the dimerization domains from both monomers in Pseudomonas sp. CPG2. Subdomains 1 (gray) and 2 (white) of PepV show apparent similarity. However, strands 8 and 12 are only found in subdomain 1, and strands 3 and 7 are only found in subdomain 2. The β-sheet composed of the latter two strands is also present in the dimerization domain of CPG2. B, backbone trace superposition of subdomains 1 and 2 in the lid domain of PepV (blue) and the two associated dimerization domains in CPG2 (red and green). Known active site residues in PepV are shown in a stick representation, from left to right, Arg350, Asn217 (both carboxyl-terminal docking), and His269 (transition state stabilization). Corresponding residues from CPG2 are also shown. The enlargement above additionally shows the corresponding residues in PepT. Arg288 from CPG2 (red) and Arg280 from PepT (yellow) reside in the monomer, which superimposes with subdomain 1 of PepV. Asn275 and His229 from CPG2 (green) and His223 in PepT (purple) are recruited from the opposite monomer which superimposes with subdomain 2 of PepV. In the structure of CPG2, the side chain of His229 shows a χ1 rotation by about 90° relative to the other two structures and coordinates an additional interdimeric zinc ion in the protein crystal (not shown). C, multiple sequence alignment of the small domains in the PepV enzymes from L. delbrueckii (PEPV_LACDL) and Lactococcus lactis subsp. cremoris MG1363 (PEPV_LACLC) and from CPG2 (CBPG_PSES6), PepT (PEPT_SALTY), and hAcy1 (ACY1_HUMAN). Subdomain 1 and 2 in the lid domain of PepV are abbreviated sd1 and sd2, respectively. The alignment was assembled using an available alignment of the two PepV enzymes (15Hellendoorn M.A. Franke-Fayard B.M. Mierau I. Venema G. Kok J. J. Bacteriol. 1997; 179: 3410-3415Crossref PubMed Google Scholar) and structure-based alignments of CPG2 to sd1 in L. Delbrueckii (29Jozic D. Bourenkow G. Bartunik H. Scholze H. Dive V. Henrich B. Huber R. Bode W. Maskos K. Structure. 2002; 10: 1097-1106Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and CPG2 to PepT (24Hakansson K. Miller C.G. Eur. J. Biochem. 2002; 269: 443-450Crossref PubMed Scopus (45) Google Scholar). The sequences of the dimerization domain in hAcy1 and CPG2 were aligned manually. Strands (s) and helices (h), as identified in the crystal structures of PepV, CPG2, and PepT, are printed in red and blue, respectively. Their numbering in sd1 and sd2 of L. delbrueckii PepV is indicated in the corresponding colors above the aligned sequences. Residues that interact with the bound transition state analog AspΨ[PO2CH2]AlaOH in the PepV structure are in yellow boxes. Greek letters indicate the sites of rearrangement generated by the insertion of sd2 in the sequence of sd1 and their sequel.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Structure of the Zinc-binding Domain in hAcy1—During crystallization screening of different mutants of hAcy1, which are under investigation in our laboratory, only the T347G mutant gave crystals. Degradation of an intervening sequence, from residue 199 to 320, corresponding to the dimerization domain was observed, and only the large zinc-binding domain remained. The degradation occurred only after the protein was concentrated to 5–8 mg/ml for the crystallization trials. The intact T347G mutant only showed a 6.5-fold increase in [S]0.5 and a reduction of Tm by 7 °C (Table V), indicating that the mutation did not affect the catalytic machinery.Table VKinetic parameters for the hydrolysis of Nα-acteyl-l-methionine and stability parameters of wild-type and mutant hAcy1 enzymeshAcy1 variantk cat[S]0.5Hill coefficient (n)ΔG(H2O)Tms-1mmkJ·mol-1°CWild type38.3 ± 0.80.43 ± 0.031.16 ± 0.0718.7 ± 0.1064T347G46.0 ± 0.772.78 ± 0.111.28 ± 0.04NDaND, not determined.57E147AbNo cooperativity was observed, and [S]0.5 = KM and n = 1.(10 ± 0.3)·10-31.06 ± 0.11118.0 ± 0.5061E147QbNo cooperativity was observed, and [S]0.5 = KM and n = 1.(4.43 ± 0.11)·10-30.71 ± 0.06113.2 ± 0.1064E147DNMcNM, not measurable.NMNMH80A (Zn2)bNo cooperativity was observed, and [S]0.5 = KM and n = 1.(15.89 ± 0.75)·10-32.02 ± 0.30114.8 ± 0.2445D113A (Zn1/2)bNo cooperativity was obser
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