Active-site-specific inhibitors

Search the Enzyme Structure Database for y-chymotrypsin active site (by the aid of the active-site-modified enzyme or active-site-specific inhibitor-enzyme complex) to identify and depict (save pdb file) the catalytic triad of y-chymotrypsin. 

Proteolytic Enzymes and Their Active-Site-Specific Inhibitors Role in the Treatment of Disease... 

Apart from lAPs, there are several nonmammalian regulators of caspases, which are active-site specific inhibitors (Callus and Vaux, 2007). One example is a serpin from the cowpox virus, cytokine response modifier A (crmA). CrmA forms a covalent complex with the initiator caspase-1 and -8 resulting in irreversible inhibition of these caspases. It also inhibits caspase-6 but less efficiently (Dobo et al., 2006). The baculoviral protein p35 is a broad spectrum caspase inhibitor that irreversibly inactivates caspases (Bump et al., 1995 Fisher et al., 1999). 

Information relevant to the mechanism of an enzyme-catalyzed reaction can, in general, only be obtained from irreversible inhibitors which react specifically at the active site and thereby inactivate the enzyme. As active-site-directed inhibition is treated in detail in Ref. 142 general aspects will be discussed here only briefly. In order to be suitable as an active-site-directed inhibitor, a compound must fulfil the following requirements. 

The active-site-directed inhibitor tosylphenylalanine chloromethyl ketone that specifically and irreversibly inhibits chymotrypsin. This chloroketone inhibitor relies on its toluene sulfonyl (or tosyl) group for binding into the aromatic binding pocket of chymotrypsin s active site. Inactivation occurs by alkylation of histidine-57 (pseudo-first order rate constant 0.2 min ). See Chymo-trypsin... 

The two proteinase Inhibitors that accumulate In leaves of wounded tomato leaves have been Isolated and characterized. Inhibitor I has a molecular weight of 41,000 and Is composed of subunits with molecular weights of about 8100 (10). It Is, therefore, a pentamer In Its native state. Each subunit possesses an active site specific for chymotrypsln, and the apparent for the Inhibition of chymotrypsln Is about 10 M (10). Inhibitor II has a molecular weight of about 23,000, Is composed of two subunits, and strongly Inhibits both trypsin and chymotrypsln with Kj values of about 10 and 10 M respectively... 

Active-site directed inhibitors have reactivity with the enzyme greatly enhanced over that of non-specific inhibitors thus phenacyl iodide inhibits papain 50-fold faster than iodoacetamide whereas the active-site directed inhibitor 4-toluenesulphonylamidomethyl chloromethyl ketone reacts some 650-fold faster. The enhanced rate is due to complexation of the inhibitor with the enzyme, and indicates that the inhibitor must be reacting at the active site. 

Fan JQ, Ishii S. Active-site-specific chaperone therapy for Fabry disease. Yin and Yang of enzyme inhibitors. FEES J. 2007 274 4962-4971. 

The crystal structure of BACE was recently determined by Tang and colleagues [270], In Figure 5.5, we show the substrate binding site from a structure alignment of the model and the crystal structure. A number of the substrate-specificity-determining residues were correctly determined both in location and conformation. In this crystal structure, an inhibitor is bound in the active site. This inhibitor has sequence EVNL-AAEF, where the L-A sequence has a non-peptide backbone. The PI position is occupied by an... 

M Lackmann, et al. Radioimmunoassay for the detection of active site specific thrombin inhibitors in biological fluids—Heparin affects the binding of hirudin to alpha-thrombin. Thromb Res 63 609, 1991. 

The strict specificity of PFL for pyruvate and formate as substrates places some restrictions on the structure of potential active site-directed inhibitors. Therefore, acetyl phosphinate (207) is a natural candidate for a PFL inhibitor, as it is not only a pyruvate analog, but is also a derivative of hypophosphite, a known mechanism-based PFL inactivator. In the absence of CoA, 100 mM acetyl phosphinate is an effective time-dependent inhibitor of PFL, although the inactivation reaction does not appear to be first order. If 55 mM CoA is included in the inactivation reaction mixture, first-order kinetics are now observed, and with 10 mM acetyl phosphinate the half-life of the inactivation reaction is 3 min. In the presence of 5 mM pyruvate (no CoA), PFL is completely protected from inactivation by 100 mM acetyl phosphinate. 

Suicide inhibitors, or mechanism-based inhibitors are modified substrates that provide the most specific means to modify an enzyme active site. The inhibitor binds to the enzyme as a substrate and is initially processed by the normal catalytic mechanism. The mechanism of catalysis then generates a chemically reactive intermediate that inactivates the enzyme through covalent modification. The fact that the enzyme participates in its own irreversible inhibition strongly suggests that the covalently modified group on the enzyme is catalytically vital. One example of such an inhibitor is N,N-dimethylpropargylamine. A flavin prosthetic group of monoamine oxidase... 

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