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Structural Characterization of the Catalytic Active Site in the Latent and Active Natural Gelatinase B from Human Neutrophils

2000; Elsevier BV; Volume: 275; Issue: 44 Linguagem: Inglês

10.1074/jbc.m005714200

1083-351X

Oded Kleifeld, Philippe E. Van den Steen, Anatoly I. Frenkel, Feng Cheng, Hua Jiang, Ghislain Opdenakker, Irit Sagi,

Peptidase Inhibition and Analysis

Matrix metalloproteinases are endopeptidases that have a leading role in the catabolism of the macromolecular components of the extracellular matrix in a variety of normal and pathological processes. Human gelatinase B is a zinc-dependent proteinase and a member of the matrix metalloproteinase family that is involved in inflammation, tissue remodeling, and cancer. We have conducted x-ray absorption spectroscopy, atomic emission, and quantum mechanics studies of natural and activated human gelatinase B. Our results show that the natural enzyme contains one catalytic zinc ion that is central to catalysis. In addition, upon enzyme activation, the catalytic zinc site exhibits a conformation change that results in the expansion of the bond distances around the zinc ion and the replacement of one sulfur with oxygen. Interestingly, quantum mechanics calculations show that oxygen ligation at the catalytic zinc ion exhibits a greater affinity to the binding of an oxygen from an amino acid residue rather than from an external water molecule. These results suggest that the catalytic zinc ion plays a key role in both substrate binding and catalysis. Matrix metalloproteinases are endopeptidases that have a leading role in the catabolism of the macromolecular components of the extracellular matrix in a variety of normal and pathological processes. Human gelatinase B is a zinc-dependent proteinase and a member of the matrix metalloproteinase family that is involved in inflammation, tissue remodeling, and cancer. We have conducted x-ray absorption spectroscopy, atomic emission, and quantum mechanics studies of natural and activated human gelatinase B. Our results show that the natural enzyme contains one catalytic zinc ion that is central to catalysis. In addition, upon enzyme activation, the catalytic zinc site exhibits a conformation change that results in the expansion of the bond distances around the zinc ion and the replacement of one sulfur with oxygen. Interestingly, quantum mechanics calculations show that oxygen ligation at the catalytic zinc ion exhibits a greater affinity to the binding of an oxygen from an amino acid residue rather than from an external water molecule. These results suggest that the catalytic zinc ion plays a key role in both substrate binding and catalysis. matrix metalloproteinases x-ray absorption spectroscopy density functional theory inductively coupled plasma atomic emission spectroscopy extended x-ray absorption fine structure x-ray absorption, near edge structure Remodeling of the extracellular matrix is an important event in many normal and pathological processes such as growth, wound repair, tumor metastasis, and leukocyte mobilization in inflammation. A large family of zinc-dependent proteinases, the matrix metalloproteinases (MMPs),1is considered to be primarily responsible for this matrix catabolism (1Birkedal-Hansen H. Curr. Opin. Cell Biol. 1995; 7: 728-735Crossref PubMed Scopus (975) Google Scholar, 2Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (701) Google Scholar, 3Kiyama R. Tamura Y. Watanabe F. Tsuzuki H. Ohtani M. Yodo M. J. Med. Chem. 1999; 42: 1723-1738Crossref PubMed Scopus (101) Google Scholar, 4Matrisian L.M. Trends Genet. 1990; 6: 121-125Abstract Full Text PDF PubMed Scopus (1531) Google Scholar). Details of their variety and substrate specificity are documented in several recent reviews (5Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar, 6Shapiro S.D. Curr. Opin. Cell Biol. 1998; 10: 602-608Crossref PubMed Scopus (622) Google Scholar, 7Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3887) Google Scholar). The MMPs consist of a large family of proteinases that share many common structural and functional elements. MMPs can be categorized based on amino acid sequence similarity, substrate specificity, and domain structure. They include collagenases, which cleave triple helical interstitial collagens; gelatinases, which cleave denatured collagen, elastin and type IV and V collagens; stromelysins, which mainly cleave proteoglycans; and membrane-type MMPs, which are associated with the activation of pro-MMPs (2Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (701) Google Scholar, 3Kiyama R. Tamura Y. Watanabe F. Tsuzuki H. Ohtani M. Yodo M. J. Med. Chem. 1999; 42: 1723-1738Crossref PubMed Scopus (101) Google Scholar). In addition to these classical MMPs, other metalloproteinases, mainly with functions other than matrix catabolism, are being discovered at an increasing pace. All MMPs are synthesized as preproenzymes and are usually secreted as inactive pro-MMPs. The primary structure of each MMP is composed of some of the following domain motifs: a signal peptide, a propeptide, a catalytic domain that contains a zinc ion central to catalysis, a linker, a hemopexin-like domain, a fibronectin type II domain, a transmembrane region and a cytoplasmic domain (found in membrane-types 1–6), a furin-recognition sequence, and a vitronectin-like domain (7Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3887) Google Scholar). Moreover, each member of the MMP family is characterized by a different domain composition, but contains a propeptide domain of about 80 amino acids with a conserved PRCGVPDV motive that ligates to the catalytic zinc ion via the cysteine residue to maintain the latency of the proenzyme (8Van Wart H.E. Birkedal-Hansen H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5582Crossref PubMed Scopus (1203) Google Scholar, 9Becker J.W. Marcy A.I. Rokosz L.L. Axel M.G. Burbaum J.J. Fitzgerald P.M. Cameron P.M. Esser C.K. Hermes J.D. Springer J.P. Protein Sci. 1995; 4: 1966-1976Crossref PubMed Scopus (271) Google Scholar). The catalytic domain (about 170 amino acids) contains a zinc-binding motive HEXXHXXGXXH, and all MMPs except MMP-7 have a hemopexin-like domain (about 210 amino acids) at the carboxyl terminus. The hemopexin-like domain is reported to be required for the digestion of native collagen by collagenase-1, -2, and -3 as well as for the binding of TIMP-1 to progelatinase B and TIMP-2 to progelatinase A (10Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). The first x-ray crystal structures of the recombinant catalytic domains of human fibroblast collagenase/MMP-1 (11Lovejoy B. Cleasby A. Hassell A.M. Longley K. Luther M.A. Weigl D. McGeehan G. McElroy A.B. Drewry D. Lambert M.H. Jordan S.R. Science. 1994; 263: 375-377Crossref PubMed Scopus (306) Google Scholar) and human neutrophil collagenase/MMP8 (12Bode W. Reinemer P. Huber R. Kleine T. Schnierer S. Tschesche H. EMBO J. 1994; 13: 1263-1269Crossref PubMed Scopus (323) Google Scholar) were soon complemented by additional structures of catalytic domains, full-length recombinant enzymes, and inhibitor-enzyme complex structures (9Becker J.W. Marcy A.I. Rokosz L.L. Axel M.G. Burbaum J.J. Fitzgerald P.M. Cameron P.M. Esser C.K. Hermes J.D. Springer J.P. Protein Sci. 1995; 4: 1966-1976Crossref PubMed Scopus (271) Google Scholar, 13Lovejoy B. Hassell A.M. Luther M.A. Weigl D. Jordan S.R. Biochemistry. 1994; 33: 8207-8217Crossref PubMed Scopus (153) Google Scholar, 14Gomis-Ruth F.X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (513) Google Scholar, 15Grams F. Crimmin M. Hinnes L. Huxley P. Pieper M. Tschesche H. Bode W. Biochemistry. 1995; 34: 14012-14020Crossref PubMed Scopus (166) Google Scholar, 16Li J. Brick P., MC, O.H. Skarzynski T. Lloyd L.F. Curry V.A. Clark I.M. Bigg H.F. Hazleman B.L. Cawston T.E. Blow D.M. Structure. 1995; 3: 541-549Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 17Bode W. Fernandez-Catalan C. Tschesche H. Grams F. Nagase H. Maskos K. Cell Mol. Life Sci. 1999; 55: 639-652Crossref PubMed Scopus (298) Google Scholar). Importantly, these detailed atomic resolution structures have revealed the location of the conserved motifs and have enabled the assignment of substrate recognition sites on the proteinases. The catalytic domains of the MMPs have an ellipsoid shape with a small active site cleft on their surfaces. This cleft contains the catalytic zinc atom. Several studies have suggested that the catalytic domain of the isolated MMPs contains two zinc atoms (11Lovejoy B. Cleasby A. Hassell A.M. Longley K. Luther M.A. Weigl D. McGeehan G. McElroy A.B. Drewry D. Lambert M.H. Jordan S.R. Science. 1994; 263: 375-377Crossref PubMed Scopus (306) Google Scholar, 18Soler D. Nomizu T. Brown W.E. Chen M. Ye Q.-Z. Van-Wart H.E. Auld D.S. Biochem. Cell Biol. 1994; 201: 917-923Google Scholar, 19Salow S. Marcy A.L. Cuca G.C. Smith C.K. Kopka I.E. Hagmann W.K. Hermes J.D. Biochemistry. 1992; 31: 4535-4540Crossref PubMed Scopus (75) Google Scholar) that are required for enzyme stabilization and catalysis. To date, however, none of these structural studies have dealt with natural or intact MMP-9. Recent reports and our results (reported here) suggest that the intact full-length MMPs contain only a single zinc atom. Together, these discrepant results suggest that the second zinc-binding site may result from a molecular rearrangement and subsequent structural stabilization due to truncation of the polypeptide chain (20Henning B. Curr. Opin. Cell Biol. 1995; 7: 725-728Google Scholar, 21Willenbrock F. Murphy G. Phillips I.R. Brocklehurst K. FEBS Lett. 1995; 358: 189-192Crossref PubMed Scopus (25) Google Scholar). The crystal and NMR structures of the various MMPs provide a framework for the design of mechanistic studies. However, the actual mechanism of MMP catalysis has yet to be elucidated. Previous studies led to the formulation of the “cysteine switch hypothesis” as a model for understanding the unique structure of MMP zymogens and the means by which activation may be achieved in vitro (8Van Wart H.E. Birkedal-Hansen H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5582Crossref PubMed Scopus (1203) Google Scholar). Briefly, the cysteine switch model suggests that upon activation, the latent zinc-binding site is converted to a catalytic zinc-binding site by dissociation of the thiol-bearing propeptide from the zinc atom. Cleavage of the propeptide results in a breakdown of the prodomain structure of the enzyme, and the shielding of the catalytic zinc ion is withdrawn. Consequently, the metal ion and the active site pocket are accessible for substrate binding (22Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Crossref PubMed Scopus (484) Google Scholar). Human gelatinase B is a matrix metalloprotease (MMP-9) involved in inflammation, tissue remodeling, development, and cancer (23Dubois B. Opdenakker G. Carton H. Acta Neurol. Belg. 1999; 99: 53-56PubMed Google Scholar, 24Masure S. Proost P. Van Damme J. Opdenakker G. Eur. J. Biochem. 1991; 198: 391-398Crossref PubMed Scopus (249) Google Scholar). Importantly, this MMP is a marker of inflammatory diseases, including rheumatoid arthritis and multiple sclerosis (25Opdenakker G. Masure S. Grillet B. Van Damme J. Lymphokine Cytokine Res. 1991; 10: 317-324PubMed Google Scholar, 26Gijbels K. Masure S. Carton H. Opdenakker G. J. Neuroimmunol. 1992; 41: 29-34Abstract Full Text PDF PubMed Scopus (285) Google Scholar). In the central nervous system, gelatinase B may play a role in the enzymatic degradation of the blood-brain barrier and may generate autoimmune peptides from intact myelin constituents, suggesting that it may be at the basis of autoimmunity in multiple sclerosis (27Opdenakker G. Van Damme J. Immunol. Today. 1994; 15: 103-104Abstract Full Text PDF PubMed Scopus (201) Google Scholar). By analogy with other proteases (28Liotta L.A. Rao C.N. Barsky S.H. Lab. Invest. 1983; 49: 636-649PubMed Google Scholar), it has also been suggested that the secretion of gelatinase B may play a key role in tumor cell metastasis by providing the basis for the mechanism of extracellular matrix remodeling (29Rao J. Steck P.A. Mohanam S. Stetler-Stevenson W. Liotta L.A. Sawaya R. Cancer Res. 1993; 53: 2208-2211PubMed Google Scholar,30Coussens L.M. Werb Z. Chem. Biol. 1996; 3: 895-904Abstract Full Text PDF PubMed Scopus (503) Google Scholar). Human neutrophils produce three major forms of pro-gelatinase B as follows: 92-kDa monomers, homodimers, and complexes of gelatinase B covalently bound to neutrophil gelatinase B-associated lipocalin (31Rudd P. Mattu T.S. Masure S. Bratt T. Van den Steen P. Wornald M.R. Kustner B. Harvery D.J. Borregaard N. Dwek R. Opdenakker G. Biochemistry. 1999; 38: 13937-13950Crossref PubMed Scopus (90) Google Scholar). Gelatinase B is not produced constitutively by most cells, but instead its activity is induced by different stimuli depending on the cell type, thus providing a means of increasing the local concentration of gelatinolytic activity in response to specific physiological events. Therefore, it is considered to be one of the most complex MMPs in terms of regulation and expression, in addition to possessing a complex domain structure (23Dubois B. Opdenakker G. Carton H. Acta Neurol. Belg. 1999; 99: 53-56PubMed Google Scholar, 24Masure S. Proost P. Van Damme J. Opdenakker G. Eur. J. Biochem. 1991; 198: 391-398Crossref PubMed Scopus (249) Google Scholar, 31Rudd P. Mattu T.S. Masure S. Bratt T. Van den Steen P. Wornald M.R. Kustner B. Harvery D.J. Borregaard N. Dwek R. Opdenakker G. Biochemistry. 1999; 38: 13937-13950Crossref PubMed Scopus (90) Google Scholar, 32Norga K. Grillet B. Masure S. Paemen L. Opdenakker G. Clin. Rheumatol. 1996; 15: 31-34Crossref PubMed Scopus (12) Google Scholar, 33Van den Steen P. Rudd P.M. Proost P. Martens E. Paemen L. Kuster B. van Damme J. Dwek R.A. Opdenakker G. Biochim. Biophys. Acta. 1998; 1425: 587-598Crossref PubMed Scopus (27) Google Scholar). Recently it was reported that glycosylation of the gelatinase B molecule is considerable in terms of molecular volume, but the function of this post-translational modification is as yet not clear (31Rudd P. Mattu T.S. Masure S. Bratt T. Van den Steen P. Wornald M.R. Kustner B. Harvery D.J. Borregaard N. Dwek R. Opdenakker G. Biochemistry. 1999; 38: 13937-13950Crossref PubMed Scopus (90) Google Scholar). Therefore, further structural analysis of the catalytic site of the full-length natural human gelatinase B may contribute to understanding its mode of action. Here we report on our x-ray absorption spectroscopy (XAS) studies of the catalytic active site of natural human gelatinase B in its latent and activated states. The XAS results are consistent with the density functional theory (DFT) calculations on the active sites of MMPs in latent and activated forms, which are also reported here. Our results demonstrate the molecular rearrangement and conformational changes that occur upon activation in human gelatinase B at the zinc site. In addition, we show by independent experimental procedures that natural human gelatinase B contains one zinc atom. Natural gelatinase B was isolated from human neutrophils by using a method modified from Masure et al. (24Masure S. Proost P. Van Damme J. Opdenakker G. Eur. J. Biochem. 1991; 198: 391-398Crossref PubMed Scopus (249) Google Scholar). Human neutrophils were isolated from buffy coats (Red Cross, Antwerp, Belgium) (34Wuyts A. Govaerts C. Struyf S. Lenaerts J.-P. Put W. Conings R. Proost P. Van Damme J. Eur. J. Biochem. 1999; 260: 421-429Crossref PubMed Scopus (75) Google Scholar). After a 20-min preincubation with 3 mmphenylmethylsulfonyl fluoride at 37 °C, 0.5 μmformyl-methionyl-leucyl-phenylalanine and 3 mmphenylmethylsulfonyl fluoride were added to stimulate degranulation for the next 20 min. The cell supernatants were harvested, filtered, loaded on a gelatin-Sepharose substrate affinity column (Amersham Pharmacia Biotech), and eluted as described (24Masure S. Proost P. Van Damme J. Opdenakker G. Eur. J. Biochem. 1991; 198: 391-398Crossref PubMed Scopus (249) Google Scholar). The eluate was then dialyzed against assay buffer (100 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10 mm CaCl2, 0.01% Tween 20). In the next step, the covalent heterodimeric complex of gelatinase B with neutrophil gelatinase B-associated lipocalin was removed by affinity chromatography on a monoclonal antibody directed against neutrophil gelatinase B-associated lipocalin, courtesy of Dr. T. Bratt and Dr. N. Borregaard (35Strong R.K. Bratt T. Cowland J.B. Borregaard N. Wiberg F.C. Ewald A.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 93-95Crossref PubMed Scopus (29) Google Scholar). The purity of gelatinase B was controlled by reducing and non-reducing SDS-polyacrylamide gel electrophoresis and analyzed by Coomassie Brilliant Blue protein staining. Protein concentration was determined using a Bradford reagent (Bio-Rad) (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216334) Google Scholar) with bovine serum albumin as a standard and according to amino acid composition analysis. The active site of gelatinase B was titrated using different concentrations of recombinant human TIMP-1 (Calbiochem) in an enzyme activity assay with a fluorogenic peptide, as described by Knight et al. (37Knight C.G. Willenbrock F. Murphy G. FEBS Lett. 1992; 296: 263-266Crossref PubMed Scopus (678) Google Scholar). The enzyme samples and the appropriate controls were analyzed by gelatin substrate zymography (38Masure S. Billiau A. Van Damme J. Opdenakker G. Biochim. Biophys. Acta. 1990; 1054: 317-325Crossref PubMed Scopus (76) Google Scholar). This technique provides information about the molecular size and the catalytic activity of the enzyme. Gelatinase B activity was measured and quantified by the conversion of a fluorogenic peptide (37Knight C.G. Willenbrock F. Murphy G. FEBS Lett. 1992; 296: 263-266Crossref PubMed Scopus (678) Google Scholar). The metal content in gelatinase B samples was analyzed by inductively coupled plasma atomic emission spectroscopy using the ICP-AES model “Spectroflame” from Spectro (Kleve, Germany). Prior to measurement, the samples were digested with nitric acid, and the volume was adjusted to 6 ml (final concentration 10%). The zinc content in the protein samples was determined relative to an equivalent amount of gelatinase assay buffer. Enzymatic activity was checked for all samples by zymography and by the conversion of the fluorogenic peptide before XAS data collection. The enzyme was concentrated by ultrafiltration using a Millipore Centricon-30 (Bedford, MA) apparatus to make a final concentration of 70 μm (6.4 mg/ml). Samples were loaded into copper sample holders (10 × 5 × 0.5 mm) covered with Mylar tape and were frozen immediately in liquid nitrogen. The frozen samples were then mounted inside a Displex closed cycle helium cryostat and their temperature was maintained at 30 K to minimize the thermal disorder in the XAS data. XAS data collection was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, beam line X9B. The spectra were recorded at the zinc K-edge in fluorescence geometry at low temperature (30 K). The beam energy was defined using a flat silicon(111) monochromator crystal. The incident beam intensity I0 was recorded using an ionization chamber. The fluorescence intensity was recorded using a 13-element germanium detector. The transmission signal from a zinc foil was measured with a reference ion chamber simultaneously with fluorescence in order to calibrate the beam energy. Several scans of each sample were collected for a total of 1 × 106 counts across the edge. The samples were checked for burning marks after each scan, and the beam position on the sample was changed before each scan to minimize radiation damages. Enzyme activity was checked after exposure to x-rays, and the enzyme was found to be fully active. The average zinc K-edge absorption coefficient μ(E), which was obtained after several independent XAS measurements for each sample, was aligned in absolute energy, using the reference zinc metal foil XAS data as an absolute energy calibrant. Subsequently, the absorption coefficients for different samples were shifted in x-ray energy until their first inflection points were aligned at the same energy (9658 eV). This alignment ensured, to a good approximation, that the same x-ray energy,E 0 = 9658 eV could be used as the photoelectron energy origin in all data sets. The smooth atomic background was removed with the AUTOBK program of the UWXAFS data analysis package, developed at the University of Washington, Seattle (39Stern E.A. Newville M. Ravel B. Yacoby Y. Haskel D. Phys. Rev. B. 1995; 208/209: 117-122Crossref Scopus (921) Google Scholar). The same energy, E 0 = 9658 eV, was chosen for the purpose of background removal as the origin of the photoelectron energy. The R space region for minimizing the signal below the first shell was chosen between 1 and 1.2 Å. After the removal of background, the useful k range in the resultant k 2 weighted χ(k) was between 2.5 and 9 Å−1. Model data for the fitting procedure were constructed by extracting the catalytic zinc site coordinates (in a radius of 6 Å) from the crystallographic coordinates of stromelysin-1 (MMP-3) derived from the protein data bank (code 1SLM) (9Becker J.W. Marcy A.I. Rokosz L.L. Axel M.G. Burbaum J.J. Fitzgerald P.M. Cameron P.M. Esser C.K. Hermes J.D. Springer J.P. Protein Sci. 1995; 4: 1966-1976Crossref PubMed Scopus (271) Google Scholar), gelatinase A (protein data bank code 1CK7), and the optimized active site structures obtained from our quantum mechanical calculations. By using the computer code FEFF7 (41Rehr J.J. Mustre, de leon J. Zabinsky S.I. Albers R.C. J. Am. Chem. Soc. 1991; 113: 5135-5138Crossref Scopus (1823) Google Scholar, 42Zabinsky S.I. Rehr J.J. Ankudinov A. Albers R.C. Eller M.J. Phys. Rev. B. 1995; 52: 2995-2999Crossref PubMed Scopus (2688) Google Scholar), we calculated the theoretical photoelectron scattering amplitudes and phase shifts. Total theoretical χ(k) was constructed by adding the most important partial χ(k)s that contributed to ther range of interest. The theoretical XAFS signal was fitted to the experimental data using the non-linear least squares method, implemented in the program FEFFIT (39Stern E.A. Newville M. Ravel B. Yacoby Y. Haskel D. Phys. Rev. B. 1995; 208/209: 117-122Crossref Scopus (921) Google Scholar) in R space, by Fourier transforming both theory and data. Data and theory were weighted by k and multiplied by a Hanning window function in Fourier transforms. The DFT computational method B3LYP was applied on a model of the first coordination shell around the catalytic zinc ion in the MMPs. The B3LYP/3-21G method was employed for full optimization of the initial model structures, and the optimized structures were then subjected to further optimization with B3LYP/6-31G*. Based on the optimized geometries, the frequency calculations were carried out at the B3LYP/6-31G* level of theory in order to verify the accuracy of the optimized structures and to determine the zero point and vibrational energies, enthalpy, and entropy parameters. All quantum mechanic calculations were carried out with the Gaussian98 program (44Frisch, M. G., Trucks, G. W., H. B., Schlegel, G. E., Scuseria, M. A., Robb, J. R., Cheeseman, V. G., Zakrzewski, J. A., Montgomery, R. E., Jr., Stratmann, J. C., Burant, S., Dapprich, J. M., Millam, A. D., Daniels, K. N., Kudin, M. C., Strain, O., Farkas, J., Tomasi, V., Barone, M., et al. (1998) Gaussian, Inc., Pittsburgh, PA.Google Scholar). The active site structures of the catalytic zinc ion in natural gelatinase B from neutrophils, in its latent and activated states, were studied by x-ray absorption, near edge structure (XANES), and extended x-ray absorption fine structure (EXAFS) spectroscopy. EXAFS is a valuable technique for elucidating the structure of a variety of metal sites in metalloproteins (45Scott R.A. Methods Enzymol. 1985; 117: 414-458Crossref Scopus (189) Google Scholar). More specifically, EXAFS measures the transition from the core electronic states of the metal to the excited electronic or continuum states. Spectral analysis near the electronic transition (XANES) provides information about the charge state of the metal and its geometry. Spectral analysis above the absorption edge, in the EXAFS region, provides complementary structural information such as coordination numbers, types, and distances from neighboring atoms to the central (absorbing) atom. In addition, XAS is an excellent structural tool to probe the d10 zinc ion, which is generally spectroscopically silent (46Kleifeld O. Frenkel A. Bogin O. Eisenstein M. Brumfeld V. Burstein Y. Sagi I. Biochemistry. 2000; 39: 7702-7711Crossref PubMed Scopus (28) Google Scholar). We therefore conducted XAS studies to examine the nature and bonding of active site ligands and the changes in metal site structures upon activation of human gelatinase B. Furthermore, EXAFS analysis was used to examine the zinc stoichiometry in human gelatinase B. For fixed geometry of the fluorescence EXAFS experiment, the edge step of the x-ray absorption coefficient should be proportional to the concentration of the absorbing element. Therefore, the number of zinc atoms per protein can be determined by comparing the edge-step intensity measured in the enzyme absorption coefficient data with the calibration curve obtained for standard compounds, where the edge step is measured as a function of the zinc concentration. Aqueous solutions in the range of 0.5–1.5 mm of ZnCl2were chosen as standard compounds for the purpose of edge intensity calibration. The calibration curve presented in Fig.1 shows a linear relationship between the edge- step intensity changes and the function of increasing zinc ion concentration (linearity in the lower concentrations of the calibration curve was obtained by linear extrapolation to zero). The enzyme concentration was independently obtained to be 0.07 mm by conducting an amino acid analysis experiment, a standard Bradford assay, and by titration of the active enzyme with TIMP-1 (see “Experimental Procedures”). To obtain the estimated concentration of zinc ion in the enzyme as measured by XAS, we crossed the experimental edge-step intensity value of gelatinase B with the calibration curve. The concentration of zinc ion obtained by this type of analysis was 0.065 mm which is consistent with one zinc atom per enzyme (Fig. 1). These results were repeated using different protein preparation batches and different germanium detectors in measuring the XAS. Since the edge-step analysis may deviate from linearity because of detector limitation at very low concentrations, we further examined the zinc stoichiometry by ICP-AES.Figure 1Edge-step analysis of natural human gelatinase B. The edge intensity of human gelatinase B is compared with the standard calibration curve. Proteins and standards were measured and processed with identical procedures. The zinc:enzyme ratio is consistent with 1:1 stoichiometry.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The atomic absorption signal of the enzyme was measured and placed on a standard calibration curve, which was obtained by measuring standards of known zinc concentrations in the range of 0–1000 parts/billion. The zinc content of the buffer solution was 2 parts/billion. The enzyme was diluted by a factor of 240 before measuring the atomic absorption. The zinc concentration in the enzyme was 17 parts/billion, which is equivalent to 62 μm in the zinc concentration (the enzyme concentration was 70 μm as described). These results show that the zinc content in the enzyme is consistent with one zinc ion per enzyme in a ratio of 0.89:1 zinc to protein and were found to be in good agreement with our edge-step analysis. The presence of single as opposed to multiple zinc sites simplifies the EXAFS data analysis procedure, since the curve-fitting analysis of the local environment around the multiple zinc ions requires an excessive number of structural variables in the analysis. However, we further examined the possibility of the contribution of multiple zinc atoms by non-linear curve-fitting EXAFS analysis to be detailed. EXAFS analysis of human gelatinase B, in its latent and activated forms, was conducted by fitting the data to theoretical phase shifts and amplitudes. Theoretical models of the proposed structural and catalytic zinc sites were constructed from the crystal structure of MMP-3 (9Becker J.W. Marcy A.I. Rokosz L.L. Axel M.G. Burbaum J.J. Fitzgerald P.M. Cameron P.M. Esser C.K. Hermes J.D. Springer J.P. Protein Sci. 1995; 4: 1966-1976Crossref PubMed Scopus (271) Google Scholar), gelatinase A (MMP-2) (22Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Crossref PubMed Scopus (484) Google Scholar), and our calculated model (see Table II). The architecture of the catalytic domain, known as the matrixin fold (47Stocker W. Bode W. Curr. Opin. Struct. Biol. 1995; 5: 383-390Crossref PubMed Scopus (189) Google Scholar), is highly conserved in MMPs and is unaffected by insertion of the fibronectin domains (22Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Crossref PubMed Scopus (484) Google Scholar). The vicinity of the binding sites of the catalytic and the structural zin