Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-08-02
2002-06-11
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S196000, C424S094600
Reexamination Certificate
active
06403330
ABSTRACT:
The invention relates to crystalline form of TRAP (tartrate-resistant and purple acid phosphatases), which is activated by cleavage. The TRAP is preferably human or rat TRAP. The atomic structural coordinates are given as well as the conserved surface which is created by special atoms, relevant for the design of modulators or inhibitors of the human TRAP. This crystalline form of activated TRAP can be used for structure-based drug design for specific modulator, activator or inhibitor of TRAP activity, useful in the treatment of diseases or degenerative conditions resulting in increased bone resorption, such as tissue damages, bone metabolic disorders, osteoporosis.
BACKGROUND
Tartrate resistant purple acid phosphatase (TRAP or PAP) is a mammalian di-iron containing enzyme highly expressed in a limited number of tissues. In humans and rodents it is primarily present in cells responsible for bone resorption, osteoclasts, and in macrophages of spleen, liver and lung.
Normal bone function requires a turnover of bone. Bone is constantly being rebuilt by cycles of resorption and formation which means that formation is closely linked to resorption (a phenomenon referred to as coupling).
TRAP is an enzyme expressed predominantly in bone resorbing cells (osteoclasts). Investigations in TRAP knockout mice show that the resorption process is disrupted so that, with increasing age, TRAP knockout mice become osteopetrotic, i.e. have an increased bone mineral content and more dense bone is formed. Osteoclasts prepared from these animals are functional and do resorb bone but to a lesser extent than wild type mouse osteoclasts.
Phosphatases are enzymes that remove organic phosphates from proteins. The mammalian Purple Acid Phosphatases (PAPs), a group of enzymes to which Tartrate Resistant and purple Acid Phosphatase (TRAP) belongs, are characterized by a binuclear iron center at the active site.
The binuclear iron center, low pH optimum (≈5), high isoelectric point (≈9) and insensitivity to inhibition by L(+) tartrate are features of TRAP that may be involved in the apparent substrate specificity at the low pH in the osteoclastic resorption area. The TRAP enzyme is a cationic glycoprotein with a molecular mass of 35 kD. The rat TRAP is a protein with a monomeric 306 amino acid peptide structure. See FIG.
1
. The peptide sequence of rat bone TRAP displays 89-94% homology to TRAP enzyme of the human placenta, bovine spleen, and uteroferrin.
TRAP hydrolyzes aryl phosphates, nucleoside di- and triphosphates, pyrophosphate and phosphoproteins. Its physiological role remains unclear but TRAP may mediate dephosphorylation of bone matrix proteins such as osteopontin and bone sialoprotein. Dephosphorylation of bone matrix proteins enables osteoclasts to migrate over the bone surface and TRAP is therefore likely to be involved in the attachment of osteoclasts to the bone surface.
In humans and rats, PAP enzymes are highly expressed in certain cells of the monocytemacrophage lineage, such as the bone-resorbing osteoclasts and certain activated macrophages in spleen, liver and lung [1-4], and TRAP has since long been used as a histochemical marker for these cells. Given the broad substrate specificity of PAP enzymes, it is conceivable that other factors, such as local availability and proper compartmentalisation of PAPs with their potential substrates, are other important factors in determining the physiological action of PAPs in biological systems.
The cDNA sequences of TRAP/PAP enzymes from different species and organs all indicate that these enzymes are translated as a single polypeptide of around 35 kDa [5-8]. This contrasts with the predominantly two subunit structure, consisting of a 20-23 kDa N-terminal domain linked through a disulphide bond to a 15-17 kDa C-terminal domain, observed in purified enzyme preparations from a variety of sources including human and rat bone [9-10], giant cell tumors [11] and normal and pathological spleen [12-14]. In contrast, uteroferrin purified from endometrial secretions are mostly in the single subunit form [12, 15] as are the recombinant PAPs generated by overexpression using the Baculovirus system [16, 17, 18]. Orlando et al [13] managed to separate the monomeric and two-subunit variants of PAP from bovine spleen, and demonstrated a markedly higher specific enzyme activity associated with the two subunit form. Moreover, digestion of the single subunit form with the serine proteases trypsin or chymotrypsin generated the 23 kDa and 15 kDa disulfide-linked fragments characteristic of the two subunit form together with a significant enhancement of enzyme activity. Similar nicking and activation of the non-cleaved purified recombinant human and mouse PAPs were noted upon prolonged storage [17].
Purple acid phosphatases (PAPs) are acid metallohydrolases that contain a binuclear Fe3+M2+ center in their active site, where M=Fe or Zn [19-22]. In mammals, these enzymes are also referred to as tartrate-resistant acid phosphatases (TRAPs) (EC 3.1.3.2) or type 5 acid phosphatases[23]. TRAPs are iron-containing, monomeric glycoproteins with molecular weights of around 35,000 Da [24]. The deduced amino acid sequences of human, rat and mouse TRAPs shows a high degree of identity to the mammalian members of the PAP family, e.g uteroferrin (Uf) and bovine spleen PAP[5-7]. Recently, EPR spectroscopic analysis of rat recombinant TRAPs[16] have provided compelling evidence that this enzyme belong to the purple acid phosphatase family.
Mammalian PAPs contain a FeFe centre, while a plant PAP from red kidney beans (KBPAP) instead has a FeZn center [25]. The anti-ferromagnetically spin-coupled binuclear iron centre of the mammalian PAPs exists in two stable interconvertible states: pink, reduced, EPR-visible and enzymatically active, with a mixed-valent Fe2+-Fe3+ cluster; and purple, oxidized, EPR-silent and catalytically inactive, with the binuclear pair as Fe3+-Fe3+ [21, 26-27]. In contrast, the plant enzyme with a mixed-valent Zn2+-Fe3+ centre is constitutively active [28 ]. The M(2+) site in the PAPs can harbour either Zn2+ or Fe2+ without alteration of enzyme activity or spectral properties [28-30]. KBPAP is the only PAP whose X-ray structure has been determined. [17] The active site of KBPAP consists of an iron and a zinc ion bridged by an aspartate and probably a hydroxide. The Fe3+ site is coordinated by tyrosine, histidine and aspartate, while the Zn2+ site is coordinated by two histidines and an asparagine [17,31]. One solvent molecule is probably bound to each metal ion. Kidney bean PAP is a homodimeric protein with a molecular weight of around 110,000 Da, and exhibits a low overall sequence homology to the mammalian PAPs [32]. However, an alignment of the sequences of Uf and KBPAP displays an identical positioning of the amino acid residues ligating the di-metal centre [31,32]. Moreover, the mammalian protein phosphatases calcineurin (type 2B) [1-2] and protein phosphatase type
1
(PP-1) [3-4] both contain a di-nuclear metal centre and also reveal a striking similarity to the plant PAP enzyme in the coordination environments of the active site, except for the absence of the tyrosine ligand. These two latter enzymes are serine/threonine protein phosphatases, suggesting that also PAPs function as protein phosphatases. A sequence motif, DXH(X)nGDXXD(X)nGNHD/E, incorporating most of the metal-coordinating amino acids found in the PAP and PP structures so far identified has recently been identified also in a large group of phosphoesterases, including other phosphomonoesterases, nucleotide phosphatases and nucleases, from plants, bacteria and animal cells [8-11]. This phosphoesterase signature motif is represented at the secondary structure level as
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Meller Mike
Naff David M.
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