Enzymes activation-inactivation

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

Fructose bisphosphate aldolase of animal muscle is a Class I aldolase, which forms a Schiff base or imme intermediate between the substrate (fructose-1,6-bisP or dihydroxyacetone-P) and a lysine amino group at the enzyme active site. The chemical evidence for this intermediate comes from studies with the aldolase and the reducing agent sodium borohydride, NaBH4. Incubation of fructose bisphosphate aldolase with dihydroxyacetone-P and NaBH4 inactivates the enzyme. Interestingly, no inactivation is observed if NaBH4 is added to the enzyme in the absence of substrate. 

An example for proteases are the (3-lactamases that hydrolyse a peptide bond in the essential (3-lactam ring of penicillins, cephalosporins, carbapenems and monobac-tams and, thereby, iireversibly inactivate the diug. 13-lactamases share this mechanism with the penicillin binding proteins (PBPs), which are essential enzymes catalyzing the biosynthesis of the bacterial cell wall. In contrast to the PBPs which irreversibly bind (3-lactams to the active site serine, the analogous complex of the diug with (3-lactamases is rapidly hydrolyzed regenerating the enzyme for inactivation of additional (3-lactam molecules. 

Figure 21-6. Regulation of acetyl-CoA carboxylase by phosphorylation/dephosphorylation.The enzyme is inactivated by phosphorylation by AMP-activated protein kinase (AMPK), which in turn is phosphorylated and activated by AMP-activated protein kinase kinase (AMPKK). Glucagon (and epinephrine), after increasing cAMP, activate this latter enzyme via cAMP-dependent protein kinase. The kinase kinase enzyme is also believed to be activated by acyl-CoA. Insulin activates acetyl-CoA carboxylase, probably through an "activator" protein and an insulin-stimulated protein kinase.

Figure 6. Maceration of potato tuber tissue pre-incubated for 2 h with 0,05 U/ml of PL3 enzyme activity. Con = Control incubated with the inactivated enzyme.

PG activity was assayed from cells grown in a medium containing 1% glucose in one-litre self-induced anaerobic fermentation for 5 days by increase of reducing sugars. Enzyme activity increased from pH 3.0 to pH 5.0 (citrate buffer) and decreased drastically above 5.0 (phosphate buffer), but activity was not affected differentially by the two buffers used (data not shown). PG activity increased almost linearly from 20°C to 40°C but above this optimum, activity was lost rapidly and the enzyme was completely inactivated at 60°C and 70°C after 10 and 6 min, respectively (data not shown). No PL, PGL or PME were detected. 

Study of the temperature optimum of pectinesterase activity showed, that peak of pectinesterase activity was observed at the temperature equal to 45 °C. It is shown on Figure 1 that pectinesterase was stable at pH 4 — 5. At pH 2 activity of the enzyme reduced by 25% in 60 min., at pH 3 and pH 6 it decreased by 6 — 8%. At pH 8 the activity decreased by 90.7% during the same time. At pH 9 the enzyme activity was inactivated during 15 min. 

A large number of amino acid transporters have been detected by isolating mutations which selectively inactivate one permease without altering enzyme activities involving the corresponding amino acid. Competitive inhibition, kinetics and regulatory behaviour have also been used as criteria to distinguish one transport system from another (see section 4.2). 

In contrast to the effects obtained with viruses mentioned earlier, rous sarcoma virus (RSV) is inactivated by direct contact with 2 [81]. Evidence for the drug action by a chelate compound was obtained by using concentrations of 3a and copper(II) sulfate, neither of which individually affected enzyme activity or transforming abilities [82]. In a later study these workers showed that several metal complexes inhibit the RNA dependent DNA polymerases and the transforming ability of RSV, the most active compound being a 1 1 copper(II)... 

All soil metabolic proce.sses are driven by enzymes. The main sources of enzymes in soil are roots, animals, and microorganisms the last are considered to be the most important (49). Once enzymes are produced and excreted from microbial cells or from root cells, they face harsh conditions most may be rapidly decomposed by organisms (50), part may be adsorbed onto soil organomineral colloids and possibly protected against microbial degradation (51), and a minor portion may stand active in soil solution (52). The fraction of extracellular enzyme activity of soil, which is not denaturated and/or inactivated through interactions with soil fabric (51), is called naturally stabilized or immobilized. Moreover, it has been hypothesized that immobilized enzymes have a peculiar behavior, for they might not require cofactors for their catalysis. 

E I is a kinetic chimera Kj and kt are the constants characterizing the inactivation process kt is the first-order rate constant for inactivation at infinite inhibitor concentration and K, is the counterpart of the Michaelis constant. The k,/K, ratio is an index of the inhibitory potency. The parameters K, and k, are determined by analyzing the data obtained by using the incubation method or the progress curve method. In the incubation method, the pseudo-first-order constants /cobs are determined from the slopes of the semilogarithmic plots of remaining enzyme activity... 

However, diffusion of the reactive QM out of the enzyme active site is a major concern. For instance, a 2-acyloxy-5-nitrobenzylchloride does not modify any nucleophilic residue located within the enzyme active site but becomes attached to a tryptophan residue proximal to the active site of chymotrypsin or papain.23,24 The lack of inactivation could also be due to other factors the unmasked QM being poorly electrophilic, active site residues not being nucleophilic enough, or the covalent adduct being unstable. Cyclized acyloxybenzyl molecules of type a could well overcome the diffusion problem. They will retain both the electrophilic hydroxybenzyl species b, and then the tethered QM, in the active site throughout the lifetime of the acyl-enzyme (Scheme 11.1). This reasoning led us to synthesize functionalized... 

With a 3,3-heterodihalogeno substitution of the (3-lactam ring, a selective interaction of each enantiomer of the chiral azetidinone with the enzyme active site is expected. The enantiomer 3R of the 3F, 3Br derivative indeed has a more favorable kinetic parameter k-JK, than the enantiomer 3S.33 The partition ratio kCA /kt (=k3/k4, Eq. 11.1) for the inactivation is also higher. Therefore, enantiomer 3R is a better suicide substrate for HLE since a lower partition ratio corresponds to abetter suicide substrate.20... 

Some nonreactive molecules are recognized by the target enzyme as pseudosubstrates. These bind to the enzyme active site and are chemically transformed into reactive species that then covalently inactivate the enzyme. 

Because these mechanism-based inactivators rely on the chemistry of the enzyme active site, they are often highly selective for the target enzyme and thus provide the specificity required for use as drug molecules. 

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