Enzyme inactivation

Suicide Substrates —Mechanism-Based Enzyme Inactivators... 

Several drugs in current medical use are mechanism-based enzyme inactivators. Eor example, the antibiotic penicillin exerts its effects by covalently reacting with an essential serine residue in the active site of glycoprotein peptidase, an enzyme that acts to cross-link the peptidoglycan chains during synthesis of bacterial cell walls (Eigure 14.17). Once cell wall synthesis is blocked, the bacterial cells are very susceptible to rupture by osmotic lysis, and bacterial growth is halted. 

Silverman, R. B., 1988. Mechanism-Based Enzyme Inactivation Chemistry and Enzymology, Vols. I and II. Boca Raton, FL CRC Press. 

In some cases, impurities in the ionic liquids resulted in dramatic pH shifts, causing enzyme inactivation. This could sometimes be overcome simply by titration or higher buffer concentrations. In other cases, purification of the ionic liquid or an improved synthesis might be necessary. 

Some enzymes require metal ions - such as cobalt, manganese or zinc - for their activity if these are removed by the ionic liquid by complexation, enzyme inactivation may occur. 

Further examples of the utility of the allylic sulfoxide-sulfenate interconversion in the construction of various biologically active natural products include intermediates such as the /Miydroxy-a-methylene-y-butyrolactones (e.g. 63)128 and tetrahydrochromanone derivative 64129. Interestingly, the facility and efficiency of this rearrangement has also attracted attention beyond the conventional boundaries of organic chemistry. Thus, a study on mechanism-based enzyme inactivation using an allyl sulfoxide-sulfenate rearrangement has also been published130 131. 

A new type of mechanism-based enzyme-inactivators, which are related to conduritol epoxides with respect to activation at the active site, was introduced by Tong and Ganem, " who prepared the aziridine 37 from the o-galacto analog of 1-deoxynojirimycin. Compound 37 proved to be a pp-... 

Breeze A.S. Simpson A.M. (1982) An improved method using acetyl-coenzyme A regeneration for the enzymic inactivation of aminoglycosides prior to sterility testing. JApplBacteriol, 53, 277-284. 

DEVECE C, RODRIGUEZ LOPEZ J N, FENOLL L G, TUDELA J, CATALA J M, DE LOS REYES E and GARCIA CANOVAS F (1999) Enzyme inactivation analysis for industrial blanching applications comparison of microwave, conventional, and combination heat treatments on mushroom polyphenoloxidase activity , J g-nc Food Chem, 47 (11) 4506-11. 

Preparation of AIR and extraction of pectic fractions For the preparation of the alcohol-insoluble residue (AIR) the apples were peeled, cut into small pieces and boiled in 96% ethanol for lOmin. After this enzyme inactivation step, the sample material was blended, homogenised and filtered through a G3 sintered glass niter funnel. The residue was washed with 96% ethanol, followed by acetone and diethyl-ether, dried overnight at 40°C under vacuum and stored at -20°C in the dark. Portions of about lOg of AIR were fractionated according to the method of Selvendran et al. [10] as shown in figure 1. 

The lyophililized intestinal contents or faeces were treated for enzyme inactivation in 5 ml 96 % EtOH for 20 min at 75-80 °C. After addition of 5 ml water the mixture was stirred 30 min and centrifuged at 6000 g also for 30 min. In the supernatant galacturonan was estimated by the m-hydroxydiphenyl (MHDP) reaction [7] and OligoGalA were determined using HPTLC. In the dried residues, the content of galacturonan and the DE were estimated after extraction with 0.5 % EDTA. 

Walsh, C. T. Suicide substrates mechanism-based enzyme inactivators recent developments. Ann. Rev. Biochem. 1984, 53, 493-535. 

Green tea processing is designed to achieve a dry product exhibiting the desirable twisted leaf appearance, but without flavanol oxidation. This is accomplished by carrying out rapid enzyme inactivation either with steam in a rotating cylinder as practiced in Japan or with dry heat as practiced in the People s Republic of China. The inactivated tea is cooled, rolled partially dried, rerolled, and then completely dried. 

Until now our discussions of enzyme inhibition have dealt with compounds that interact with binding pockets on the enzyme molecule through reversible forces. Hence inhibition by these compounds is always reversed by dissociation of the inhibitor from the binary enzyme-inhibitor complex. Even for very tight binding inhibitors, the interactions that stabilize the enzyme-inhibitor complex are mediated by reversible forces, and therefore the El complex has some, nonzero rate of dissociation—even if this rate is too slow to be experimentally measured. In this chapter we turn our attention to compounds that interact with an enzyme molecule in such a way as to permanendy ablate enzyme function. We refer to such compounds as enzyme inactivators to stress the mechanistic distinctions between these molecules and reversible enzyme inhibitors. 

Figure 8.1 Typical enzyme reaction progress curve in the presence of an irreversible enzyme inactivator, highlighting the initial velocity region (v ) and the fact that the terminal velocity (vs) is zero for such compounds.

Figure 8.2 Mechanisms of irreversible enzyme inactivation. (A) Nonspecific affinity labeling, (B) quiescent affinity labeling, and (C) mechanism-based inactivation.

Hence, for any irreversible enzyme inactivator, we can quantify the effectiveness of inactivation using the second-order rate constant kanJKi. This constant thus becomes the key metric that the medicinal chemist can use in exploring the SAR of enzyme inactivation by a series of compounds. In terms of individual rate constants, the definitions of both nact and A) depend on the details of the mechanisms of inactivation, as will be described below. 

An important point to realize here is that attempts to quantify the relative potency of irreversible enzyme inactivators by more traditional parameters, such as IC50 values, are entirely inappropriate because these values will vary with time, in different ways for different compounds. Hence the SAR derived from IC50 values, determined at a fixed time point in the reaction progress curve, is meaningless and can be misleading in terms of compound optimization. Unfortunately, the literature is rife with examples of this type of inappropriate quantitation of irreversible inactivator potency, making meaningful comparisons with literature data difficult, at best. 

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