Noncovalent inhibitor-protein

Over a decade ago, work on the enzyme aldolase reductase elegantly demonstrated this point. The noncovalent inhibitor alrestatin was modified to contain various electrophiles a-chloroacetamide, a-bromoacetamide or a-iodoacetamide. Noncovalent interactions between inhibitors and protein would not have changed, but molecules behaved differently based on the electrophile the weakest showed reversible inhibition, whereas the iodoacetamide displayed almost complete irreversible inhibition.1401 These results are an important warning if a reaction is too facile, irreversible reactions can obscure true binding affinities. 

Fig. 5. Protein folding. The unfolded polypeptide chain coUapses and assembles to form simple stmctural motifs such as -sheets and a-hehces by nucleation-condensation mechanisms involving the formation of hydrogen bonds and van der Waal s interactions. Small proteins (eg, chymotrypsin inhibitor 2) attain their final (tertiary) stmcture in this way. Larger proteins and multiple protein assembhes aggregate by recognition and docking of multiple domains (eg, -barrels, a-helix bundles), often displaying positive cooperativity. Many noncovalent interactions, including hydrogen bonding, van der Waal s and electrostatic interactions, and the hydrophobic effect are exploited to create the final, compact protein assembly. Further stmctural...

The enzymatic activity of these potentially harmful enzymes is tightly controlled. Once transcribed into protein, MMPs are expressed as inactive zymogens and require distinct activation processes to convert them into active enzymes. After secretion, MMP-activity is regulated by the noncovalent binding of tissue inhibitors of metalloproteinases ( TIMPs) as shown in Fig. 2 for MMP-2 and TIMP-2. Four TIMPs have been identified so far TIMP-1, TIMP-2, TIMP-3, and TIMP-4. All known MMPs can be inhibited by at least one of the four known TIMPs. Nevertheless, individual differences with regard to bond strength and thus the magnitude of inhibition of a particular MMP do exist. 

As we have just seen, the initial encounter complex between an enzyme and its substrate is characterized by a reversible equilibrium between the binary complex and the free forms of enzyme and substrate. Hence the binary complex is stabilized through a variety of noncovalent interactions between the substrate and enzyme molecules. Likewise the majority of pharmacologically relevant enzyme inhibitors, which we will encounter in subsequent chapters, bind to their enzyme targets through a combination of noncovalent interactions. Some of the more important of these noncovalent forces for interactions between proteins (e.g., enzymes) and ligands (e.g., substrates, cofactors, and reversible inhibitors) include electrostatic interactions, hydrogen bonds, hydrophobic forces, and van der Waals forces (Copeland, 2000). 

Collectively, the direct thrombin inhibitors are prototypically represented by hirudin, the antithrombotic molecule found in the saliva of the medicinal leech (Hirudo medicinalis), This protein is a 65 amino acid molecule that forms a highly stable but noncovalent complex with thrombin (7). With two domains, the NH2-terminal core domain and the COOH-terminal tail, the hirudin molecule inhibits the catalytic site and the anion-binding exosite in a two-step process. The first step is an ionic interaction that leads to a rearrangement of the thrombin-hirudin complex to form a tighter bond that is stoi-chiometrically I I and irreversible. The apolar-binding site may also be involved in hirudin binding. This complex and... 

Figure 13 Molecular-mass analysis of phosphorylase B (panel B) and the separation of the noncovalently (Sypro Red) labeled protein markers (panel A ALA, a-lactalbumin CBA, carbonic anhydrase OVA, ovalbumin BSA, bovine serum albumin BGA, (f-galactosidase) and the covalently (FITC) labeled protein markers (panel C TRI, tripsin inhibitor CAH, carbonic anhydrase ADH, alcohol dehydrogenase BSA, bovine serum albumin BGA, P-galactosidase). Separation conditions gel, 1% agarose, 2% linear polyacrylamide (LPA, MW 700,000-1,000,000) in 50 mM Tris, 50 mM TAPS, 0.05% SDS (pH 8.4) separation buffer, 50 mM Tris, 50 mM TAPS, 0.05% SDS (pH 8.4) separation voltage, 420 V, current, 5 mA gel thickness, 190 pm effective separation length, 3.5 cm temperature, 25°C sample loading, 0.2 pL into 2.5 x 4 x 0.19-mm injection wells. Sample buffer contained 0.05% SDS and 1 x Sypro Red. (Reproduced with permission from Ref. 141.)...

Covalent bonds are not as important in drug-receptor binding as noncovalent interactions. Alkylating agents in chemotherapy tend to react and form an immonium ion, which then alkylates proteins, preventing their normal participation in cell divisions. Baker s concept of active site directed irreversible inhibitors was well established by covalent formation of Baker s antifolate and dihydrofolate reductase (46). 

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