Serine proteases comprise nearly one-third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so-called ''classical'' catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of ''nonclassical'' serine proteases where variations to the catalytic triad are observed. Such variations include the triads Ser/His/Glu, Ser/ His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine peptidases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. This work discusses the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class.
Aza-peptide Michael acceptors are a new class of irreversible inhibitors that are highly potent and specific for clan CD cysteine proteases. The aza-Asp derivatives were specific for caspases, while aza-Asn derivatives were effective legumain inhibitors. Aza-Lys and aza-Orn derivatives were potent inhibitors of gingipain K and clostripain. Aza-peptide Michael acceptors showed no cross reactivity toward papain, cathepsin B, and calpain.
Aza-peptide Michael acceptors are a novel class of inhibitors that are potent and specific for caspases-2, -3, -6, -7, -8, -9, and -10. The second-order rate constants are in the order of 10(6) M(-1) s(-1). The aza-peptide Michael acceptor inhibitor 18t (Cbz-Asp-Glu-Val-AAsp-trans-CH=CH-CON(CH(2)-1-Naphth)(2) is the most potent compound and it inhibits caspase-3 with a k(2) value of 5620000 M(-1) s(-1). The inhibitor 18t is 13700, 190, 6.4, 594, 37500, and 173-fold more selective for caspase-3 over caspases-2, -6, -7, -8, -9, and -10, respectively. Aza-peptide Michael acceptors designed with caspase specific sequences are selective and do not show any cross reactivity with clan CA cysteine proteases such as papain, cathepsin B, and calpains. High-resolution crystal structures of caspase-3 and caspase-8 in complex with aza-peptide Michael acceptor inhibitors demonstrate the nucleophilic attack on C2 and provide insight into the selectivity and potency of the inhibitors with respect to the P1' moiety.
Aza-peptide epoxides, a novel class of irreversible protease inhibitors, are specific for the clan CD cysteine proteases. Aza-peptide epoxides with an aza-Asp residue at P1 are excellent irreversible inhibitors of caspases-1, -3, -6, and -8 with second-order inhibition rates up to 1 910 000 M(-1) s(-1). In general, the order of reactivity of aza-peptide epoxides is S,S > R,R > trans > cis. Interestingly, some of the R,R epoxides while being less potent are actually more selective than the S,S epoxides. Our aza-peptide epoxides designed for caspases are stable, potent, and specific inhibitors, as they show little to no inhibition of other proteases such as the aspartyl proteases porcine pepsin, human cathepsin D, plasmepsin 2 from P. falciparum, HIV-1 protease, and the secreted aspartic proteinase 2 (SAP-2) from Candida albicans; the serine proteases granzyme B and alpha-chymotrypsin; and the cysteine proteases cathepsin B and papain (clan CA), and legumain (clan CD).
Aging is a dealkylation reaction of organophosphorus (OP)-inhibited acetylcholinesterase (AChE). Despite many studies to date, aged AChE cannot be reactivated directly by traditional pyridinium oximes. This review summarizes strategies that are potentially valuable in the treatment against aging in OP poisoning. Among them, retardation of aging seeks to lower the rate of aging through the use of AChE effectors. These drugs should be administered before AChE is completely aged. For postaging treatment, realkylation of aged AChE by appropriate alkylators may pave the way for oxime treatment by neutralizing the oxyanion at the active site of aged AChE. The other two strategies, upregulation of AChE expression and introduction of exogenous AChE, cannot resurrect aged AChE but may compensate for lowered active AChE levels by in situ production or external introduction of active AChE. Upregulation of AChE expression can be triggered by some peptides. Sources of exogenous AChE can be whole blood or purified AChE, either from human or nonhuman species.
After the inhibition of acetylcholinesterase (AChE) by organophosphorus (OP) nerve agents, a dealkylation reaction of the phosphylated serine, referred to as aging, can occur. When aged, known reactivators of OP-inhibited AChE are no longer effective. Realkylation of aged AChE may provide a route to reversing aging. We designed and synthesized a library of quinone methide precursors (QMPs) as proposed realkylators of aged AChE. Our lead compound (C8) from an in vitro screen successfully resurrected 32.7 and 20.4% of the activity of methylphosphonate-aged and isopropyl phosphate-aged electric-eel AChE, respectively, after 4 days. C8 displays properties of both resurrection (recovery from the aged to the native state) and reactivation (recovery from the inhibited to the native state). Resurrection of methylphosphonate-aged AChE by C8 was significantly pH-dependent, recovering 21% of activity at 4 mM and pH 9 after only 1 day. C8 is also effective against isopropyl phosphate-aged human AChE.
Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at ؊1 (P1) and aliphatic residues at the ؊3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the ؊1 position (Karla, Proteins destined for secretion are synthesized in a precursor form with an amino-terminal extension peptide that targets the exported protein to the Sec machinery (1) or the Tat machinery (2) in bacteria. During the export process, the signal peptide is cleaved from the precursor protein by a signal peptidase that is embedded in the plasma membrane.AIn Escherichia coli, signal peptidase (SPase I) 2 consists of a single polypeptide chain of 37 kDa (3). This enzyme spans the membrane twice with a small cytoplasmic segment (residues 29 -58) and a large carboxyl-terminal catalytic domain located in the periplasm (residues 77-323) (4 -6). Catalysis by SPase I is carried out by a Ser-Lys dyad (7-10). In the case of the E. coli SPase I, Ser-90 is the nucleophilic residue that attacks the scissile bond of the precursor substrate and lysine 145 is the general base that deprotonates the serine residue (for review, see Ref. 11). A critical serine and lysine residue is also present in SPases from other species of bacteria (12), and members of the signal peptidase I family in mitochondria (13).With the exception of the mitochondrial inner membrane peptidase I (Imp1), all type I signal peptidases carry out processing with a specificity for small aliphatic residues at the Ϫ1 (P1) and Ϫ3 (P3) positions (11). Alanine is usually the preferred amino acid residue at the Ϫ1 and Ϫ3 positions and results in the frequently observed "Ala-X-Ala" motif for signal peptide cleavage (14 -16). The residues of SPase I that comprise the substrate binding site have been identified by solving the x-ray structure of the soluble catalytic domain with a covalently attached 5S penem inhibitor (10) and a structure with a non-covalent lipohexapeptide inhibitor (17). The three-dimensional structure of SPase I with no inhibitor bound (apo-structure) revealed that there is some variation in the binding pocket volume when compared with the inhibitor-bound structures (18). The E. coli SPase residues making direct van der Waals contact with the P1 methyl group are . Those making contact with the P3 residues are Phe-84, Ile-144, Val-132, and Ile-86. The substrate binding to SPase I occurs in an extended conformation. Recently, we have made mutations of the E. coli SPase I in the S1 and S3 pockets that bind the P1 and P3 residues of the substrate to identify the residues that control the substrate specificity (19). We found that alterations of the Ile-144 and Ile-86 residues to alanine residues could alter the substrate specificity and lead to cleava...
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