Active-site-specific inhibitors proteases

Eco is a powerful tool for defining the active sites of serine protease due to the extended substrate-like interaction that it makes with the protease. The three-dimensional structure of a complex with eco has many advantages that a structure of a protease alone or bound to a small molecule inhibitor does not have. Eco can be used to take a molecular impression of the serine protease active site and reveal features that determine substrate preference. These features are used to design specific inhibitors with therapeutic prospects. Often, a small molecule inhibitor is used to define a protease active site cleft, but the resulting structures have particular drawbacks. Typically, a small molecule inhibitor lacks the prime side interac-... 

The most thoroughly studied mechanism of protein protease inhibitors is that of the standard mechanism (or Canonical or Laskowski mechanism) inhibitors of serine proteases (1) (Fig. 2). Standard mechanism inhibitors include the Kazal, Kunitz, and Bowman-Birk family of inhibitors and bind in a lock-and-key fashion. Ah standard mechanism inhibitors insert a reactive loop into the active site of the protease, which is complementary to the substrate specificity of the target protease and binds in an extended fi-sheet with the enzyme in a substrate-like manner. WhUe bound to the protease, the scissile bond of standard mechaiusm inhibitors is hydrolyzed very slowly, but products are not released and the amide bond is re-ligated. The standard mechanism is an efficient way to inhibit serine proteases, and it is thus used by many structurally... 

In the recent studies, the enzyme shows that the overall polypeptide fold of chymotrypsin-like serine protease possesses essential SI specificity determinants characteristic of elastase using the multiple isomorphous replacement (MIR) method and refined to 2.3 A resolution Fig. (5). Structure-based inhibitor modeling demonstrated that EFEa s SI specificity pocket is preferable for elastase-specific small hydrophobic PI residues, while its accommodation of long and/or bulky PI residues is also feasible if enhanced binding of the substrate and induced fit of the SI pocket are achieved [Fig. (6) shows the active sites of serine protease]. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. This structure is the first report of an earthworm fibrinolytic enzyme component, a serine protease originated from annelid worm [17]. 

Enzymological studies using various group-specific inactivators of proteases indicated that renin belongs to the class of acidic proteases. The structural requirements of renin substrates were studied to elucidate the exceptionally selective substrate specificity of this peptidase whose only known function is to generate angiotensin I. These studies have led to the design and synthesis of active site-directed inhibitors of renin. 

A class of DUBs only identified since 2002 is the OTU (ovarian tumor protein) DUB class. The OTU domain was originally identified in an ovarian tumor protein from Drosophila mdanogaster, and over 100 proteins from organisms ranging from bacteria to humans are annotated as having an OTU domain. The members of this protein superfamily were annotated as cysteine proteases, but no specific function had been demonstrated for any of these proteins. The first hint of a role for OTU proteins in the ubiquitin pathway was afforded by the observation that an OTU-domain-containing protein, HSPC263, reacted with ubiquitin vinyl sulfone (an active-site-directed irreversible inhibitor of DUBs) [41]. 

Whereas standard proteases use serine, cysteine, aspartate, or metals to cleave peptide bonds, the proteasome employs an unusual catalytic mechanism. N-terminal threonine residues are generated by self-removal of short peptide extensions from the active yS-subunits and act as nucleophiles during peptide-bond hydrolysis [23]. Given its unusual catalytic mechanism, it is not surprising that there are highly specific inhibitors of the proteasome. The fungal metabolite lactacystin and the bacterial product epoxomicin covalently modify the active-site threonines and in-... 

Selected entries from Methods in Enzymology [vol, page(s)] Active site, structure, 241, 214 catalytic mechanism, 241, 223-224 crystal structure, 241, 214, 216 comparative studies with HIV protease [catalytic properties, 241, 205-224 evolutionary relationships, 241, 196-197 screening for HIV-1 protease inhibitors, 241, 318-321 structure, 241, 254-257, 280 substrate specificity, 241, 255, 283]. 

Several inhibitor-protease complexes have been crystallized and details of their interactions are known. For example, the pancreatic trypsin inhibitor binds at the active site of trypsin with K( >1013 M-1 at neutral pH 496 Tire two molecules fit snugly together,490 497 the inhibitor being bound as if it were a peptide substrate with one edge of the inhibitor molecule forming an antiparallel (1 structure with a peptide chain in the enzyme. Lysine 15, which forms part of this P structure, enters the specific Pj binding site for a basic amino acid in a substrate. Thus, the protease inhibitor is a modified substrate which may actually undergo attack at the active site. However, the fit between the two... 

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