Active-site-directed inhibitors

If k2 > kj, the glycosyl-enzyme intermediate will accumulate, and may be trapped by the rapid denaturation of the enzyme in the presence of (saturating) amounts of substrate. With -glucoside Aj from Asp. wentii and 4-nitrophenyl [ C]-2-deoxy-) -D-irra />jo-hexopyranoside, it was possible to identify the intermediate as a glycosyl ester (acylal) of 2-deoxy-D-arabino-hexose bound to the same aspartate residue that had previously been labeled with the active-site-directed inhibitor conduritol B epoxide and with D-glucal." This constituted an important proof that the carboxylate reacting with the epoxide is directly involved in catalysis. 

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. 

Figure 6.18 Structure of HIV-1 aspartyl protease in the flap open (left panel) and flap closed conformation with an active site-directed inhibitor bound right panel). See color insert.

A. Purohit, G. J. Williams, N. M. Howarth, B. V. L. Potter, M. J. Reed, Inactivation of Steroid Sulfatase by an Active Site-Directed Inhibitor, Estrone-3-O-sulfamate , Biochemistry 1995, 34, 11508- 11514. 

Active Site Directed Inhibitor and Point Mutation. 12... 

In the previous studies using inhibitors and additives, it became clear that AMDase requires no cofactors, such as biotin, coenzyme A and ATP. It is also suggested that at least one of four cysteine residues plays an essential role in asymmetric decarboxylation. One possibility is that the free SH group of a cysteine residue activates the substrate in place of coenzyme A. Aiming at an approach to the mechanism of the new reaction, an active site-directed inhibitor was screened and its mode of interaction was studied. Also, site-directed mutagenesis of the gene coding the enzyme was performed in order to determine which Cys is located in the active site. 

Banner, D.W. and Hadvary, P. Crystallographic analysis at 3.0 angstroms resolution of the binding to human thrombin of four active site-directed inhibitors./. Biol. Chem. 1991, 266, 20085-20090. 

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... 

G. Legler and E. Bause, Epoxy alkyl oligo-( I — 4)(3-D-glucosides as active site-directed inhibitors of cellulases, Carbohydr. Res. 28 45 (1973). 

Scheme 11.18 Active-site directed inhibitors analogous to the papain substrate, N-(4-toluenesulphonyl) glycine methyl ester.

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. 

Irreversible inhibitors may be classified for convenience as active site directed inhibitors and suicide or irreversible mechanism based inhibitors (IMBIs). They bind to the enzyme by either strong non-covalent or strong covalent bonds. Inhibitors bound by strong non-covalent bonds will slowly dissociate, releasing the enzyme to carry out its normal function. However, whatever the type of binding, the enzyme will resume its normal function once the organism has synthesized a sufficient number of additional enzyme molecules to overcome the effect of the inhibitor. 

Active site directed inhibitors are compounds that bind at or near to the active site of the enzyme. These inhibitors usually form strong covalent bonds with either the functional groups that are found at the active site or close to that site. Since these groups are usually nucleophiles, the incorporation of electrophilic groups in the structure of a substrate can be used to develop new inhibitors (Table 7.3). This approach may also be used to enhance the action of a known inhibitor. Most of the active site directed irreversible inhibitors in clinical use were not developed from a substrate. They were obtained or developed by other routes and only later was their mode of action discovered. For example, aspirin,... 

Table 7.3 Examples of the electrophilic groups used to produce active site directed inhibitors...

Of special interest is the reaction of an acyl enzyme with active-site-directed inhibitors in comparison with the free enzyme. Therefore, we studied the hydrolysis of the substrate Bz-Arg-pNA by trypsin and benzoyl-trypsin in the presence of the naturally occurring inhibitor aptotinin [21]. The substrate is cleft... 

Figure 7.18 Photoactive active-site directed inhibitors of porcine pancreatic a-amylase. Three inhibitors were synthesized by Lehmann et al.,215 with (m = 1 and n = 2), (m = 2 and n = 1) and (m = 3 and n = 0).

Figure 7.19 Oligosaccharide active-site directed inhibitors for porcine pancreatic a-amylase, synthesized by Lehmann and Schmidt-Schuchardt.218...

Anatoxin-a(s) inhibits acetylcholinesterase by acting as an irreversible active-site-directed inhibitor [61]. This prevents degradation of ACh and leads to over-stimulation of the muscle cells (Figure 6.1) [56,62]. Thus, although the mechanism of action of anatoxin-a(s) is quite different from that of anatoxin-a, the observed toxicity is similar. In addition, it was the first irreversible acetylcholinesterase inhibitor to be found in a cyanobacterium. 

Stein, R., Zuhl, F., Dick, L., Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism. J. Biol. Chem. 1997, 272, 26103-26109. 

Hyde, E.G., and Carmicheal, W.W. 1991. Anatoxin-a(s), a naturally occorring organophosphate, is an irreversible active site directed inhibitor of acetylcholinesterase (EC 3.1.1.T). JBiochem Toxicol 6, 195-201. 

In the diagram, which letter represents the non-active site-directed inhibitor present curve ... 

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