Inhibition active-site-specific

Treatment of 9-(/ -D-ribofuranosyluronic acid)adenine with diphenylphosphoro-chloridate and orthophosphate or tripolyphosphate yields (62) and (63), which, although unstable, inhibit rabbit AMP aminohydrolase and pyruvate kinase, respectively, with behaviour characteristic of active-site-specific reagents.98 Adenylate kinases from several sources are inactivated by iV6-[2- and 4-fluorobenzoyl]-adenosine-5 -triphosphates, with kinetics characteristic of active-site labelling, although these compounds were without effect on yeast hexokinase and rabbit pyruvate kinase.99... 

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... 

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). 

Activity Site Specificity Site Activ itis Reduction of Inhibits Reduction of... 

A number of inhibitors directed towards the active site of PKC have been developed [4]. Many of these have therapeutic potential and some are in clinical trials. The drug enzastaurin (LY317615) shows selectivity towards inhibiting PKC 3 and is currently in clinical trials for cancer. This drug has particular potential as a treatment for colon cancer because of the specific role ofPKC (311 in this disease (see above). A separate PKC (3 inhibitor, ruboxistaurin (LY333531) has been developed as a drug to treat the microvasculature complications of diabetes hyperactivation of both PKC (311 and PKC (31 contribute to diabetic retinopathy and microvasculature complications. 

Figure 2. Mechanism of PDH. The three different subunits of the PDH complex in the mitochondrial matrix (E, pyruvate decarboxylase E2, dihydrolipoamide acyltrans-ferase Ej, dihydrolipoamide dehydrogenase) catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. E, decarboxylates pyruvate and transfers the acetyl-group to lipoamide. Lipoamide is linked to the group of a lysine residue to E2 to form a flexible chain which rotates between the active sites of E, E2, and E3. E2 then transfers the acetyl-group from lipoamide to CoASH leaving the lipoamide in the reduced form. This in turn is oxidized by E3, which is an NAD-dependent (low potential) flavoprotein, completing the catalytic cycle. PDH activity is controlled in two ways by product inhibition by NADH and acetyl-CoA formed from pyruvate (or by P-oxidation), and by inactivation by phosphorylation of Ej by a specific ATP-de-pendent protein kinase associated with the complex, or activation by dephosphorylation by a specific phosphoprotein phosphatase. The phosphatase is activated by increases in the concentration of Ca in the matrix. The combination of insulin with its cell surface receptor activates PDH by activating the phosphatase by an unknown mechanism.

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. 

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