Pronounced diversity in electronic and chemical properties between the catalytic zinc sites of tumor necrosis factor-alpha-converting enzyme and matrix metalloproteinases despite their high structural similarity

The metalloproteinase tumor necrosis factor-alpha-converting enzyme (TACE) is involved in the regulation of several key physiological and pathological processes. Therefore, potent and selective synthetic inhibitors are highly sought for the study of the physiological roles of TACE as well as for therapeutic purposes. Because of the high structural similarities between the active site of TACE and those of other related zinc endopeptidases such as disintegrin (ADAMs) and matrix metalloproteinases (MMPs), the design of such tailor-made inhibitors is not trivial. To obtain new insights into this problem, we have used a selective MMP inhibitor as a probe to examine the structural and kinetic effects occurring at the active site of TACE upon inhibition. Specifically, we used the selective MMP mechanism-based inhibitor SB-3CT to characterize the fine structural and electronic differences between the catalytic zinc ions within the active sites of TACE and MMP-2. We show that SB-3CT directly binds the metal ion of TACE as observed before with MMP-2. However, in contrast to MMP-2, the binding mode of SB-3CT to the catalytic zinc ion of TACE is different in the length of the Zn-S(SB-3CT) bond distance and the total effective charge of the catalytic zinc ion. In addition, SB-3CT inhibits TACE in a non-competitive fashion by inducing significant conformational changes in the structure. For MMP-2, SB-3CT behaved as a competitive inhibitor and no significant conformational changes were observed. An examination of the second shell amino acids surrounding the catalytic zinc ion of these enzymes indicated that the active site of TACE is more polar than that of MMP-2 and of other MMPs. On the basis of these results, we propose that although there is a seemingly high structural similarity between TACE and MMP-2, these enzymes are significantly diverse in the electronic and chemical properties within their active sites.     

Structural basis for potent slow binding inhibition of human matrix metalloproteinase-2 (MMP-2)

The zinc-dependent gelatinases belong to the family of matrix metalloproteinases (MMPs), enzymes that have been shown to play a key role in angiogenesis and tumor metastasis. These enzymes are capable of hydrolyzing extracellular matrix (ECM) components under physiological conditions. Specific and selective inhibitors aimed at blocking their activity are highly sought for use as potential therapeutic agents. We report herein on a novel mode of inhibition of gelatinase A (MMP-2) by the recently characterized inhibitors 4-(4-phenoxphenylsulfonyl)butane-1,2-dithiol (inhibitor 1) and 5-(4-phenoxphenylsulfonyl) pentane-1,2-dithiol (inhibitor 2). These synthetic inhibitors are selective for MMP-2 and MMP-9. We show that the dithiolate moiety of these inhibitors chelates the catalytic zinc ion of MMP-2 via two sulfur atoms. This mode of binding results in alternation of the coordination number of the metal ion and the induction of conformational changes at the microenvironment of the catalytic zinc ion; a set of events that is likely to be at the root of the potent slow binding inhibition behavior exhibited by these inhibitors. This study demonstrates a distinct approach for the understanding of the structural mechanism governing the molecular interactions between potent inhibitors and catalytic sites of MMPs, which may aid in the design of effective inhibitors.     

N-Hydroxyurea as zinc binding group in matrix metalloproteinase inhibition: mode of binding in a complex with MMP-8

The first crystallographic structure of an N-hydroxyurea inhibitor bound into the active site of a matrix metalloproteinase is reported. The ligand and three other analogues were prepared and studied as inhibitors of MMP-2, MMP-3, and MMP-8. The crystal structure of the complex with MMP-8 shows that the N-hydroxyurea, contrary to the analogous hydroxamate, binds the catalytic zinc ion in a monodentate rather than bidentate mode and with high out-of-plane distortion of the amide bonds.     

Flexibility and variability of TIMP binding: X-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2

The excessive activity of matrix metalloproteinases (MMPs) contributes to pathological processes such as arthritis, tumor growth and metastasis if not balanced by the tissue inhibitors of metalloproteinases (TIMPs). In arthritis, the destruction of fibrillar (type II) collagen is one of the hallmarks, with MMP-1 (collagenase-1) and MMP-13 (collagenase-3) being identified as key players in arthritic cartilage. MMP-13, furthermore, has been found in highly metastatic tumors. We have solved the 2.0 A crystal structure of the complex between the catalytic domain of human MMP-13 (cdMMP-13) and bovine TIMP-2. The overall structure resembles our previously determined MT1-MMP/TIMP-2 complex, in that the wedge-shaped TIMP-2 inserts with its edge into the entire MMP-13 active site cleft. However, the inhibitor is, according to a relative rotation of approximately 20 degrees, oriented differently relative to the proteinase. Upon TIMP binding, the catalytic zinc, the zinc-ligating side chains, the enclosing MMP loop and the S1' wall-forming segment move significantly and in concert relative to the rest of the cognate MMP, and the active site cleft constricts slightly, probably allowing a more favourable interaction between the Cys1(TIMP) alpha-amino group of the inhibitor and the catalytic zinc ion of the enzyme. Thus, this structure supports the view that the central N-terminal TIMP segment essentially defines the relative positioning of the TIMP, while the flanking edge loops determine the relative orientation, depending on the individual target MMP.     

Molecular dynamics simulations of matrix metalloproteinase 2: role of the structural metal ions

Herein we investigate the role played by the so-called "structural metal ions" in the catalytic domain of the matrix metalloproteinase 2 enzyme (MMP-2 or gelatinase A). We performed seven molecular dynamics simulations that differ in the number and position of the noncatalytic zinc and calcium ions bound to the MMP-2 catalytic domain. An additional simulation including the three fibronectin-type modules inserted into the catalytic domain was also carried out. The analysis of the trajectories confirms that the binding/removal of the structural ions does not perturb the secondary structure elements but influences the position of several solvent-exposed loop regions that are placed near the active site cleft. The position of these loops modulates the accessibility of important anchorage points for substrate binding that have been identified in the active site groove. On the basis of semiempirical quantum chemical calculations, we estimated the relative free energies of the MMP-2 models, obtaining thus that the binding of two zinc and two calcium ions to the MMP-2 catalytic domain is energetically favored. In this MMP-2 model, which shows the most compact structure, all of the substrate binding sites are readily accessible. Globally, our results help to rationalize at the atomic level the calcium and zinc dependence of the hydrolytic activity catalyzed by the MMPs.     

The canonical methionine 392 of matrix metalloproteinase 2 (gelatinase A) is not required for catalytic efficiency or structural integrity: probing the role of the methionine-turn in the metzincin metalloprotease superfamily

Matrix metalloproteinases (MMPs) are an important family of extracellular proteases that process a variety of biologically significant molecules. MMPs are members of the metzincin superfamily of >770 zinc endopeptidases, which includes astacins, serralysins, adamalysins, leishmanolysins, and snapalysins. Metzincins are characterized by an absolutely conserved methionine residue COOH-terminal to the third histidine in the consensus sequence HEXXHXXGXX(H/D), where the histidine residues chelate a catalytic zinc ion. The canonical methionine is part of a tight 1,4-beta-turn that loops the polypeptide chain beneath the catalytic zinc ion, forming a hydrophobic floor to the Zn(2+) ion binding site. The role of this methionine is uncertain, but its absolute conservation indicates an essential catalytic or structural function. To investigate this hypothesis, we replaced Met-392 that forms the Met-turn of human MMP-2 (gelatinase A) by site-directed mutagenesis. The catalytic competence of leucine and serine mutants was assessed. (M392L)MMP-2 and (M392S)MMP-2 cleaved the physiological substrates gelatin, native type I collagen, and the chemokine monocyte chemoattractant protein-3 with similar efficiency to wild-type MMP-2. These mutants also cleaved two quenched fluorescent peptide substrates with a k(cat)/K(m) comparable to wild-type MMP-2 and underwent 4-aminophenylmercuric acetate-induced autoactivation with similar kinetics. (M392L)MMP-2 and (M392S)MMP-2 were inhibited by tissue inhibitor of metalloproteinases (TIMP)-1, -2, and -4 and by the zinc chelators 1,10-phenanthroline and a synthetic hydroxamate inhibitor, Batimastat, similar to the wild-type protein, indicating an unaltered active site topography. A tryptic susceptibility assay also suggested that (M392L)MMP-2 and (M392S)MMP-2 were correctly folded. These results challenge the dogma that this methionine residue and the Met-turn, which are absolutely conserved in all of the subfamilies of the metzincins, play an essential role in catalysis or active site structure.     

Inhibition of MMP-9 by a selective gelatinase inhibitor protects neurovasculature from embolic focal cerebral ischemia

ABSTRACT: BACKGROUND: Cerebral ischemia has been shown to induce activation of matrix metalloproteinases (MMPs), particularly MMP-9, which is associated with impairment of the neurovasculature, resulting in blood-brain barrier breakdown, hemorrhage and neurodegeneration. We previously reported that the thiirane inhibitor SB-3CT, which is selective for gelatinases (MMP-2 and 9), could antagonize neuronal apoptosis after transient focal cerebral ischemia. RESULTS: Here, we used a fibrin-rich clot to occlude the middle cerebral artery (MCA) and assessed the effects of SB-3CT on the neurovasculature. Results show that neurobehavioral deficits and infarct volumes induced by embolic ischemia are comparable to those induced by the filament-occluded transient MCA model. Confocal microscopy indicated embolus-blocked brain microvasculature and neuronal cell death. Post-ischemic SB-3CT treatment attenuated infarct volume, ameliorated neurobehavioral outcomes, and antagonized the increases in levels of proform and activated MMP-9. Embolic ischemia caused degradation of the neurovascular matrix component laminin and tight-junction protein ZO-1, contraction of pericytes, and loss of lectin-positive brain microvessels. Despite the presence of the embolus, SB-3CT mitigated these outcomes and reduced hemorrhagic volumes. Interestingly, SB-3CT treatment for seven days protected against neuronal laminin degradation and protected neurons from ischemic cell death. CONCLUSION: These results demonstrate considerable promise for the thiirane class of selective gelatinase inhibitors as potential therapeutic agents in stroke therapy.     

Structural insights into the binding mode of flavonols with the active site of matrix metalloproteinase-9 through molecular docking and molecular dynamic simulations studies

Cartilage degradation in rheumatoid arthritis is mediated principally by the collagenases and gelatinases. Gelatinase B (also called matrix metalloproteinase 9 – MMP-9), is a valid target molecule which is known to participate in cartilage degradation as well as angiogenesis associated with the disease and inhibition of its activity shall prevent cartilage damage and angiogenesis. The focus of this study is to investigate the possibilities of MMP-9 inhibition by flavonol class of bioflavonoids by studying their crucial binding interactions at the active site of MMP 9 using molecular docking (Glide XP and QPLD) and further improvisation by post-docking MM-GBSA and molecular dynamic (MD) simulations. The results show that flavonols can convincingly bind to active site of MMP-9 as demonstrated by their stable interactions at the S1′ specificity pocket and favourable binding energies. Gossypin has emerged as a promising candidate with a docking score of ?14.618 kcal/mol, binding energy of ?79.97 kcal/mol and a stable MD pattern over 15 ns. In addition, interaction mechanisms with respect to catalytic site zinc are also discussed. Further, the drug-like characters of the ligands were also analysed using ADME analysis.     

Structure of recombinant mouse collagenase-3 (MMP-13).

The matrix metalloproteinases are crucial in the physiological and pathological degradation of the mammalian extracellular matrix, including breast tumours, and osteoarthritic cartilage. These enzymes are classified according to their matrix substrate specificity. Collagenase-3 (MMP-13) is a member of this family and preferentially cleaves type II collagen, cartilage, fibronectin and aggrecan. Collagenase-3 is normally expressed in hypertrophic chondrocytes, periosteal cells, and osteoblasts during bone development. The structure of the catalytic domain of recombinant mouse collagenase-3, complexed to the hydroxamate inhibitor (RS-113456), is reported at 2.0 A resolution. Molecular replacement and weak phasing information from a single derivative determined the structure. Neither molecular replacement nor derivative methods had a sufficient radius of convergence to yield a refinable structure. The structure illuminates the atomic zinc ion interactions with functional groups in the active site, emphasizing zinc ligation and the very voluminous hydrophobic P1' group for the inhibitor potency. The structure provides insight into the specificity of this enzyme, facilitating design of specific inhibitors to target various diseases.     

Crystal structure of human macrophage elastase (MMP-12) in complex with a hydroxamic acid inhibitor

Human macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) that plays, like other members of the family, an important role in inflammatory processes contributing to tissue remodelling and destruction. In particular, a prominent role of MMP-12 in the destruction of elastin in the lung alveolar wall and the pathogenesis of emphysema has been suggested. It is therefore an attractive therapeutic target. We describe here the crystal structure of the catalytic domain of MMP-12 in complex with a hydroxamic acid inhibitor, CGS27023A. MMP-12 adopts the typical MMP fold and binds a structural zinc ion and three calcium ions in addition to the catalytic zinc ion. The enzyme structure shows an ordered N terminus close to the active site that is identical in conformation with the superactivated form of MMP-8. The S1'-specificity pocket is large and extends into a channel through the protein, which puts MMP-12 into the class of MMPs 3, 8 and 13 with large and open specificity pockets. The two crystallographically independent molecules adopt different conformations of the S1'-loop and its neighbouring loop due to differing crystal packing environments, suggesting that flexibility or the possibility of structural adjustments of these loop segments are intrinsic features of the MMP-12 structure and probably a common feature for all MMPs. The inhibitor binds in a bidentate fashion to the catalytic zinc ion. Its polar groups form hydrogen bonds in a substrate-like manner with beta-strand sIV of the enzyme, while the hydrophobic substituents are either positioned on the protein surface and are solvent-exposed or fill the upper part of the specificity pocket. The present structure enables us to aid the design of potent and selective inhibitors for MMP-12.     

A model of BmK CT in inhibiting glioma cell migration via matrix metalloproteinase-2 from experimental and molecular dynamics simulation study

The significance of BmK CT, a key chlorotoxin-like peptide isolated from the scorpion venom of Buthus martensii Karsch, is a novel blocker of the chloride ion channel and matrix metalloproteinase-2 (MMP-2). Site-directed mutagenesis of BmK CT, wound healing assay, gelatin zymography assay and computational simulation highlight the importance of electrostatic contribution to BmK CT–MMP-2 catalytic domain complex and a model of BmK CT–MMP-2 catalytic domain complex is therefore proposed. This is the first documentation of the structural mechanism of in the inhibition of glioma cell migration by BmK CT and may lead to the molecular design of specific inhibitors of MMP-2.     

Tetrahydroisoquinoline-3-carboxylate based matrix-metalloproteinase inhibitors: design,synthesis and structure-activity relationship

The design, synthesis and structure-activity relationship (SAR) of a series of nonpeptidic 2-arylsulfonyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylates and-hydroxamates as inhibitors of the matrix metalloproteinase human neutrophil collagenase (MMP-8) is described here. Based on available X-ray structures of MMP-8/inhibitor complexes, our structure-based design strategy was directed to complement major protein-ligand interaction regions mainly in the S1' hydrophobic specificity pocket close to the catalytic zinc ion. Here, the rigid 1,2,3,4-tetrahydroisoquinoline scaffold (Tic) provides ideal geometry to combine hydroxamates and carboxylates as typical zinc complexing functionalities, with a broad variety of S1' directed mono- and biaryl substituents consisting of aromatic rings perfectly accommodated within this more hydrophobic region of the MMP-8 inhibitor binding site. The effect of different S1' directed substituents, zinc-complexing groups, chirality and variations of the tetrahydroisoquinoline ring-system is investigated by systematic studies. X-ray structure analyses in combination with 3D-QSAR studies provided an additional understanding of key determinants for MMP-8 affinity in this series. The hypothetical binding mode for a typical molecule as basis for our inhibitor design was found in good agreement with a 1.7 A X-ray structure of this candidate in complex with the catalytic domain of human MMP-8. After analysis of all systematic variations, 3D-QSAR and X-ray structure analysis, novel S1' directed substituents were designed and synthesized and biologically evaluated. This finally results in inhibitors, which do not only show high biological affinity for MMP-8, but also exhibit good oral bioavailability in several animal species.     

Hydroxamate-based peptide inhibitors of matrix metalloprotease 2

There is major interest in designing inhibitors for matrix metalloproteinase 2 (MMP-2, gelatinase A) since this enzyme is known to be involved in pathological processes such as tumor invasion or rheumatoid arthritis. The majority of MMP-2 inhibitor candidate drugs block the active site of MMP-2 by binding to its catalytic Zn2+ ion through a chelating (hydroxamate, sulphonate etc.) group. Despite the general interest in designing MMP-2 inhibitors, the results with many of the drug candidates were disappointing, their failure was usually explained by cross-reactions with other MMPs. One way to enhance MMP-2 selectivity is to design inhibitors that interact with both the active site and exosites such as the fibronectin type II (FN2) domains of the enzyme. In the present work, we have examined the inhibitory potential and MMP-2 selectivity of hydroxamates of three groups of peptides known to bind to the collagen-binding FN2 domains of MMP-2. The first type of peptides consisted of collagen-like (Pro-Pro-Gly)(n) repeats, peptides of the second group were identified from a random 15-mer phage display library based on their binding to immobilized FN2 domains of MMP-2. A hydroxamate of peptide p33-42, known to bind to the third FN2 domain of MMP-2 has also been tested. Our studies have shown that these compounds inhibited MMP-2 with IC50 values of 10-100 microM. The fact that their inhibitory potential was nearly identical for MMP-2del, a recombinant version of MMP-2 that lacks the FN2 domains, suggests that inhibition is not mediated by their binding to FN2 domains. It seems likely that the failure to exploit interaction with the FN2 domains is due to the fact that the FN2 domains and the catalytic domain of MMP-2 tumble independently, therefore only a tiny fraction of the conformational isomers can bind peptide hydroxamates via both the active site and the FN2 domain(s).     

Structural Insights into the Catalytic Domains of Human Matrix Metalloprotease-2 and Human Matrix Metalloprotease-9: Implications for Substrate Specificities

Structural information for the gelatinases A (MMP-2) and B (MMP-9), two members of the matrix metalloprotease (MMP) family of enzymes, has been elusive. For the first time, computational structures for the catalytic domains of MMP-2 and MMP-9 are reported herein using the program COMPOSER and the reported three-dimensional structures of the fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8) and stromelysin-1 (MMP-3). The details of the structures of the catalytic domains of gelatinases and interactions with the protein substrate are discussed. The first analysis of the extent of hydrophobicity of surfaces in the active sites of six MMPs (including the two gelatinases reported herein) is presented to provide distinction for substrate specificity among these metalloproteases. The information from the extent of hydrophobicity/hydrophilicity analysis and general topology for these MMPs was utilized in the proposal of a method for categorization of MMPs of known three-dimensional fold. These efforts provide the first information useful to experimentalists working on the biochemical properties of these important members of the MMP family of enzymes, and provide for an opportunity to compare and contrast structures of gelatinases, collagenases and stromelysins.Electronic Supplementary Material available.     

Structural Insights into the Catalytic Domains of Human Matrix Metalloprotease-2 and Human Matrix Metalloprotease-9: Implications for Substrate Specificities

Structural information for the gelatinases A (MMP-2) and B (MMP-9), two members of the matrix metalloprotease (MMP) family of enzymes, has been elusive. For the first time, computational structures for the catalytic domains of MMP-2 and MMP-9 are reported herein using the program COMPOSER and the reported three-dimensional structures of the fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8) and stromelysin-1 (MMP-3). The details of the structures of the catalytic domains of gelatinases and interactions with the protein substrate are discussed. The first analysis of the extent of hydrophobicity of surfaces in the active sites of six MMPs (including the two gelatinases reported herein) is presented to provide distinction for substrate specificity among these metalloproteases. The information from the extent of hydrophobicity/hydrophilicity analysis and general topology for these MMPs was utilized in the proposal of a method for categorization of MMPs of known three-dimensional fold. These efforts provide the first information useful to experimentalists working on the biochemical properties of these important members of the MMP family of enzymes, and provide for an opportunity to compare and contrast structures of gelatinases, collagenases and stromelysins.     

Antibodies targeting the catalytic zinc complex of activated matrix metalloproteinases show therapeutic potential

Endogenous tissue inhibitors of metalloproteinases (TIMPs) have key roles in regulating physiological and pathological cellular processes. Imitating the inhibitory molecular mechanisms of TIMPs while increasing selectivity has been a challenging but desired approach for antibody-based therapy. TIMPs use hybrid protein-protein interactions to form an energetic bond with the catalytic metal ion, as well as with enzyme surface residues. We used an innovative immunization strategy that exploits aspects of molecular mimicry to produce inhibitory antibodies that show TIMP-like binding mechanisms toward the activated forms of gelatinases (matrix metalloproteinases 2 and 9). Specifically, we immunized mice with a synthetic molecule that mimics the conserved structure of the metalloenzyme catalytic zinc-histidine complex residing within the enzyme active site. This immunization procedure yielded selective function-blocking monoclonal antibodies directed against the catalytic zinc-protein complex and enzyme surface conformational epitopes of endogenous gelatinases. The therapeutic potential of these antibodies has been demonstrated with relevant mouse models of inflammatory bowel disease. Here we propose a general experimental strategy for generating inhibitory antibodies that effectively target the in vivo activity of dysregulated metalloproteinases by mimicking the mechanism employed by TIMPs.     

Apolactoferrin inhibits the catalytic domain of matrix metalloproteinase-2 by zinc chelation.

Lactoferrin (LTF) is a multifunctional iron-binding protein that is also capable of binding other divalent metal cations, especially Zn2+. Recent investigations indicate that lactoferrin levels are elevated in many disease conditions in which matrix metalloproteinases (MMPs), particularly MMP-2, are also elevated, suggesting that the 2 proteins may interact. This possibility was examined by determining the effect of LTF in its holo (metal-bound) and apo (metal-free) forms on the proteolytic activity of MMP-2 and other similar zinc metalloproteases. Pre-incubation with apolactoferrin, but not hololactoferrin, greatly reduced the hydrolysis of a peptide substrate by MMP-2, but not by MMP-1, -8, -9, or -13. This inhibition was specific for the 42 kDa catalytic domain fragment of MMP-2 lacking the hemopexin domain, since the 66 kDa form was poorly inhibited by apolactoferrin. The inhibition of the MMP-2 catalytic domain was strongly temperature sensitive, indicating that the conformation of one or both proteins is crucial to this interaction. To ascertain the mechanism of inhibition, increasing concentrations of ZnCl2 and FeCl2 were added to the reaction. While addition of Fe2+ did not reverse inhibition, the addition of Zn2+ resulted in a recovery of MMP-2 activity, and furthermore, zinc-saturated LTF did not inhibit MMP-2. Together, these data strongly suggest that apolactoferrin is capable of removing the catalytic zinc from the active site of MMP-2, although an exosite-based interaction between the 2 proteins cannot be fully ruled out. This inhibitory activity suggests a novel function for LTF and may represent a novel regulatory mechanism that regulates proteolysis by MMP-2 in vivo.     

Thrombin-dependent MMP-2 activity is regulated by heparan sulfate

Like most metalloproteases, matrix metalloprotease 2 (MMP-2) is synthesized as a zymogen. MMP-2 propeptide plays a role in inhibition of catalytic activity through a cysteine-zinc ion pairing, disruption of which results in full enzyme activation. A variety of proteases have been shown to be involved in the activation of pro-MMP-2, including metalloproteases and serine proteases. In the previous study we showed that MMP-2 activation occurred via specific cleavages of the propeptide by thrombin followed by intermolecular autoproteolytic processing for full enzymatic activity. Thrombin also degraded MMP-2, but this degradation was reduced greatly under cell-associated conditions with a concomitant increase in activation, prompting us to elucidate the molecular mechanisms underlying thrombin-mediated MMP-2 activation. In the present study we demonstrate that heparan sulfate is essential for thrombin-mediated activation of pro-MMP-2. Binding of heparan sulfate to thrombin is primarily responsible for this activation process, presumably through conformational changes at the active site. Furthermore, interaction of MMP-2 with exosites 1 and 2 of thrombin is crucial for thrombin-mediated MMP-2 degradation, and inhibition of this interaction by heparan sulfate or hirudin fragment results in a decrease in MMP-2 degradation. Finally, we demonstrated interaction between exosite 1 and hemopexin-like domain of MMP-2, suggesting a regulatory role of hemopexin-like domain in MMP-2 degradation. Taken together, our experimental data suggest a novel regulatory mechanism of thrombin-dependent MMP-2 enzymatic activity by heparan sulfate proteoglycans.     

Spectroscopic studies of inhibited alcohol dehydrogenase from Thermoanaerobacter brockii: proposed structure for the catalytic intermediate state

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) catalyzes the reversible oxidation of secondary alcohols to the corresponding ketones using NADP(+) as the cofactor. The active site of the enzyme contains a zinc ion that is tetrahedrally coordinated by four protein residues. The enzymatic reaction leads to the formation of a ternary enzyme-cofactor-substrate complex; and catalytic hydride ion transfer is believed to take place directly between the substrate and cofactor at the ternary complex. Although crystallographic data of TbADH and other alcohol dehydrogenases as well as their complexes are available, their mode of action remains to be determined. It is firmly established that the zinc ion is essential for catalysis. However, there is no clear agreement about the coordination environment of the metal ion and the competent reaction intermediates during catalysis. We used a combination of X-ray absorption, circular dichroism (CD), and fluorescence spectroscopy, together with structural analysis and modeling studies, to investigate the ternary complexes of TbADH that are bound to a transition-state analogue inhibitor. Our structural and spectroscopic studies indicated that the coordination sphere of the catalytic zinc site in TbADH undergoes conformational changes when it binds the inhibitor and forms a pentacoordinated complex at the zinc ion. These studies provide the first active site structure of bacterial ADH bound to a substrate analogue. Here, we suggest the active site structure of the central intermediate complex and, more specifically, propose the substrate-binding site in TbADH.     

Molecular cloning and expression of gelatinases (MMP-2 and MMP-9) in the pufferfish Takifugu rubripes

To determine the metabolism location of the extra-cellular matrix proteins in fugu (Takifugu rubripes), we cloned the cDNAs of the fugu gelatinases, matrix metalloproteinase-2 (MMP-2) and MMP-9, and examined their expressions in various adult tissues using a quantitative real-time PCR. The expression profiles of fugu gelatinases were different among tissues. FgMMP-9 mRNA was abundant in tissues that contain blood cells abundantly where fgMMP-2 mRNA was little expressed. We also examined the expression of these genes in fugu embryos during development using a whole mount in situ hybridization. Fugu MMP-2 mRNA was expressed in the pharyngeal area and mesenchyme in embryos at 80 hours post fertilization (hpf). While fugu MMP-9 mRNA was expressed in the vent at 140 hpf and the caudal end of the fin fold at 172 hpf. Although fugu MMP-2 mRNA was expressed in the pectoral fin bud at 120 hpf, fugu MMP-9 mRNA did not appear in this tissue until 10 days post fertilization (dpf). These data show expression profiles differ between the fugu gelatinases and suggest expressions of these genes are controlled at the matrix protein degradation site in fugu embryos during development.