Enzymes inhibitory sites

O Figure 4-10a shows a reaction scheme for interactions of enzyme and substrate with a full noncompetitive inhibitor. The inhibitor interacts with a site distinct from the active site, and the ESI complex is incapable of yielding product. It is thus possible, at saturating concentrations of inhibitor, to drive all enzymes to a nonproductive form, and so activity can be completely inhibited. Furthermore, the affinity of the inhibitor for the saturable allosteric inhibitory site remains independent of substrate concentration. A Lineweaver-Burk plot (O Figure 4-1 Ob) reveals a common intersection point on the 1/ [ S] axis for the data obtained at different inhibitor concentrations. It can be seen that as inhibitor concentration increases toward infinity, the slope of the Lineweaver-Burk plot increases toward infinity. Thus, a replot of the slopes versus inhibitor concentrations (O Figure 4-lOc) generates a straight line, which intersects the [i] axis at a value equal to —Ki. 

However, the concentrations required for protection (10-1 M) were much higher than were required to saturate the catalytic sites and corresponded to FDP concentrations which inhibit FDPase activity (14)- The results suggest that the enzyme contains two distinct sites for FDP, one the high affinity catalytic site, and the other an inhibitory site which is occupied only at much higher concentrations of FDP. 

This geminal type of functionality occurs when the sugar moiety is in specific stereo orientation, and with acetamido functionality. Additionally, the basic functional group (-NHAc) may act as a binding site with receptors. Such disaccharides should be valuable tools to probe any enzyme inhibitory activity of synthesized (1 -2)-S-thiodisaccharides. 

As for deaminase, the kinetic analysis suggests a partial mixed-type inhibition mechanism. Both the Ki value of the inhibitor and the breakdown rate of the enzyme-substrate-inhibitor complex are dependent on the chain length of the PolyP, thus suggesting that the breakdown rate of the enzyme-substrate-inhibitor complex is regulated by the binding of Polyphosphate to a specific inhibitory site (Yoshino and Murakami, 1988). More complicated interactions were observed between PolyP and two oxidases, i.e. spermidine oxidase of soybeen seedling and bovine serum amine oxidase. PolyP competitively inhibits the activities of both enzymes, but may serve as an regulator because the amino oxydases are also active with the polyamine-PolyP complexes (Di Paolo et al., 1995). 

When working with purified enzymes, it can be useful to perform a close examination of their phosphorylation states and molecular masses. Mass spectrometry is often useful for this purpose. Post-translational modifications or sequence truncations can potentially alter the compound binding sites available and can also change the structure of potential inhibitory sites. For example, with protein kinases, phosphorylations distal from the ATP binding site can inactivate the kinase whereas phosphorylations near the ATP binding site can activate the catalytic activity. Often, practice does not permit control of such situations because the purified systems are often mixtures and cannot be controlled in the commonly used recombinant expression technologies. 

In addition to being an inhibitor of papain-like cysteine proteases, cystatin C has recently been shown be an efficient inhibitor of some of the cysteine proteases of another family of cysteine proteases, called the peptidase family C13, with human legumain as a typical enzyme (C6). Human legumain has, like cathepsin S, been proposed to be involved in the class n MHC presentation of antigens (M3). It has also been shown that the cystatin C inhibitory site for mammalian legumain does not overlap with the cystatin C inhibitory site for papain-like cysteine proteases (Fig. 1) and that the same cystatin C molecule therefore is able to simultaneously inhibit one cysteine protease of each type (A 10). 

Although the biochemical functions and the stereochemistry of the enzyme active sites are completely established, and many natural and synthetic compounds have been assayed for their inhibitory activity, there is no an unique or preferred structural t5q>e for the highest effectiveness. In this review we have classified the active compounds in phenolics, terpenoids, alkaloids, and other principles. 

Penicillin G acylase (PGA) has pivotal role in industry for the synthesis of penicillin antibiotics. PGA catalyzes the hydrolysis of peniciUm and other P-lactam antibiotics to produce 6-amino penicillinic acid [53, 54]. Stability of PGA was investigated by assaying the enzyme activity at different time points after incubation of PGA in various ILs [53]. In the absence of substrate, about 2,000-fold increase in t, was observed in a hydrophobic IL, [EMIM][Tf2N] with respect to twpropanol. Whereas in the presence of substrate, PGA showed less stability in [Tf N]" containing ILs. The reverse trend was found for PGA in a water miscible IL [BMIM][PFg], i.e., PGA showed 9 times increase in tj in [BMIM][PF ] in the presence of substrate even at an elevated temperature of 40°C [54]. These altered t, values in hydrophobic ILs in the presence of substrates were explained on the basis of cumulative outcome of specific interaction of substrates with the active site of enzyme and inhibitory effect of the hydrolytic products formed in the reaction medium. It can be speculated that in a more hydrophilic IL like [BMIM][PF ], substrates shield the enzyme active site from direct interaction with the ionic matrix and thus imparts a stabilizing effect whereas in hydrophobic IL [EMIM][TfjN], the inhibitory effect of hydrolytic products predominates and destabilizes the enzyme. 

Na+ compared to K+, since Na+ binds to a phosphate monoanion and K+ binds to a dianion. Recent T1+-NMR data indicates this monovalent cation binds 4.0 A from the enzyme bound Mn2+ and that this distance increases to 5,4 A on binding of phosphate consistent with the mechanism of Fig. 8 (81). However, the possibility of T1+ binding at an inhibitory site rather than its activating site has not been excluded. 

Enolase catalyzes the trans dehydration of 2-phosphoglycerate to yield phosphoenolpyruvate and water only a small free energy change ( 1 kcal/mol) is associated with the reaction. The process is entropically driven and is readily reversible. This dimeric protein requires a divalent cation for activity and is rather promiscuous in that any one of about nine different cations can activate the enzyme (86). Depending upon the cation studied, the apoenzyme has either one or two metal binding sites per subunit. Metal ions such as Mg + and Mn + have one site per monomer, whereas Co " and Zn + will bind at two sites. In the presence of substrate there are two sites per subunit for all of the metal ions and, depending upon the pH, a third site is also induced. As the pH decreases, the third site is lost but not sites I and II. This third site is an inhibitory site, as the loss of this site parallels the loss of metal ion inhibition (87). The nature of this inhibition is not clear but may be due to the binding of the substrate at the phos-... 

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