[15] Reversible enzyme inhibition

In this paper, we perform a complete analysis of the kinetic behavior of the general modifier mechanism of Botts and Morales in both equilibrium steady states and non-equilibrium steady states (NESS). Enlightened by the non-equilibrium theory of Markov chains, we introduce the net flux into discussion and acquire an expression of the rate of product formation in NESS, which has clear biophysical significance. Up till now, it is a general belief that being an activator or an inhibitor is an intrinsic property of the modifier. However, we reveal that this traditional point of view is based on the equilibrium assumption. A modifier may no longer be an overall activator or inhibitor when the reaction system is not in equilibrium. Based on the regulation of enzyme activity by the modifier concentration, we classify the kinetic behavior of the modifier into three categories, which are named hyperbolic behavior, bell-shaped behavior, and switching behavior, respectively. We show that the switching phenomenon, in which a modifier may convert between an activator and an inhibitor when the modifier concentration varies, occurs only in NESS. Effects of drugs on the Pgp ATPase activity, where drugs may convert from activators to inhibitors with the increase of the drug concentration, are taken as a typical example to demonstrate the occurrence of the switching phenomenon.

3,3′-Diindolylmethane (DIM), derived from indole-3-carbinol (I3C), is used as a dietary supplement for its putative anticancer effects that include suppression of mammary tumor growth in female rats. The mechanism of action DIM may involve its interaction(s) with hepatic cytochromes P450 (CYPs) catalyzing oxidations of 17β-estradiol (E2). Our study showed that DIM added to hepatic microsomes of female Sprague–Dawley rats was primarily a competitive inhibitor of β-naphthoflavone (β-NF)- or I3C-induced CYP1A1 probe activity, and a potent mixed or uncompetitive inhibitor of phenobarbital (PB)-induced CYP2B1 or CYP2B2 probe activity, respectively. Microsomal metabolites of DIM were tentatively identified as two mono-hydroxy isomers of DIM, each formed preferentially by CYP1A1- or CYP2B1/2-catalyzed reaction. Evaluation of the effects of co-treatment of rats with PB and DIM by a full factorial ANOVA showed that DIM decreased the PB-induced CYP2B1 and CYP2B2 mRNA expression levels, and the rates of 2- and 4-hydroxylation of E2, and total E2 metabolite formation. The results suggest that interactions of DIM, and/or its mono-hydroxy metabolites, with CYP2B1 and CYP2B2 found to occur in hepatic microsomes upon addition of DIM or co-treatment of rats with DIM affect the rates of relevant oxidations of E2, and potentially protect against estrogen-dependent tumorigenesis.

Suicide substrates are widely used in enzymology for studying enzyme mechanisms and designing potential drugs. The presence of a reversible modifier decreases or increases the rate of substrate-induced inactivation, with evident physiological and experimental consequences. To date, only the action of a competitive or uncompetitive inhibitor of an enzyme system involving suicide substrate has been reported. In this paper, we analyse the kinetics of enzyme-catalysed reactions which evolve in accordance with the general modifier mechanisms of Botts and Morales in which enzyme inactivation is induced by suicide substrate. Rapid equilibrium of all of the reversible reaction steps involved is assumed and the time course equations for the residual enzyme activity, the inactive enzyme forms and the reaction product are derived. Partition ratios giving the relative weight of the product and inactive enzyme concentrations, and the relative contribution to the product formation of each of the unmodified and modified catalytic routes, are studied. New indices pointing to the conditions under which the modifier acts as inhibitor or as activator are suggested. The goodness of the analytical solutions is tested by comparison with the simulated curves obtained by numerical integration. An experimental design and kinetic data analysis to evaluate the kinetic parameters from the time progress curves of the product are proposed. From these results, those corresponding to several reaction mechanisms involving both a suicide substrate and a modifier, and which can be regarded as particular cases of the general case analysed here, can be directly and easily derived.

A phospholipase A₂ inhibitor has been purified from the serum of Notechis ater using DEAE-Sephacel chromatography. The inhibitor was found to be composed of two protein subunits (α and β) that form the intact complex of approximately 110 kDa. The α-chain is a 30-kDa glycoprotein and the β-chain a nonglycosylated, 25-kDa protein. N-terminal sequence analysis reveals a high level of homology to other snake phospholipase A₂ inhibitors. The inhibitor was shown to be extremely pH and temperature stable. The inhibitor was tested against a wide variety of phospholipase A₂ enzymes and inhibited the enzymatic activity of all phospholipase A₂ enzymes tested, binding with micromole to nanomole affinity. Furthermore, the inhibitor was compared with the Eli-Lilly compound LY311727 and found to have a higher affinity for human secretory nonpancreatic phospholipase A₂ than this chemical inhibitor. The role of the carbohydrate moiety was investigated and found not to affect thein vitro function of the inhibitor.

A combined rational and library approach was used to identify bisphosphonates (IC₅₀=20 μM) and galactose type 1-N-iminosugar (IC₅₀=45 μM) as novel motifs for selective inhibition of β-1,4-galactosyltransferase (β-1,4-GalT) and α-1,3-galactosyltransferase (α-1,3-GalT), respectively. Our results demonstrate that, though these two galactosyltransferases both utilize the same donor sugar-nucleotide (UDP-Gal), the difference in their mechanisms can be utilized to design donor sugar or nucleotide analogues with inhibitory activities selective for only one of the galactosyltransferases. Investigation of β-1,4-GalT inhibition using UDP-2-deoxy-2-fluorogalactose (UDP-2-F-Gal), UDP, and bisphosphonates, also led to the observation of metal dependent inhibition of β-1,4-GalT. These observations and the novel inhibitor motifs identified in this study pave the way for the design and identification of even more potent and selective galactosyltransferase inhibitors. ©

The impact of a sublethal concentration of an organochlorine pesticide endosulfan on the activity, specific activity, electrophoretic patterns and kinetic properties of crude and purified forms of cytoplasmic malate dehydrogenase (cMDH) and mitochondrial malate dehydrogenase (mMDH) was evaluated in the liver of the freshwater catfish, Clarias batrachus. The endosulfan reduced significantly the activity and the specific activity of cMDH and mMDH, but had no effect on total cytoplasmic and mitochondrial protein contents. The inhibition produced by endosulfan was of mixed non-competitive-uncompetitive type (K_iE > K_iES) and of mixed competitive-non-competitive type (K_iE < K_iES) for crude cMDH and mMDH, respectively. The PAGE shows five distinct isoforms (C1 to C5) of cMDH and two isoforms (M1 and M2) of mMDH. The C5-isoform of liver cMDH is predominant and it corresponds to M2-isoform of mMDH. There are no endosulfan-associated differences in the relative charges of crude cMDH and mMDH as well as their purified isoforms, C5-cMDH and M2-mMDH. The relative molecular weights of the purified isoforms are not affected by endosulfan. The purified C5-cMDH and M2-mMDH of endosulfan-treated liver in vivo showed simple non-competitive (K_iE = K_iES) type of inhibition; whereas in vitro it was of uncompetitive (K_iES) and mixed competitive-non-competitive (K_i < K_iES) type for the two respective isoforms. G-1-P acts as an uncompetitive (K_iES) inhibitor of C5-cMDH and mixed competitive-non-competitive (K_iE < K_iES) inhibitor of M2-mMDH of the control fish. The inhibitory pattern of G-1-P is modulated by endosulfan in case of C5-cMDH; whereas there is no alteration in case of M2-mMDH. Summarizing, it can be stated that endosulfan exerts an inhibitory effect on crude cMDH and mMDH in vivo, and their purified isoforms (C5-cMDH and M2-mMDH) in vivo as well as in vitro. The impact of endosulfan is mediated through enzyme-substrate-endosulfan (ES-END) complexing for cMDH and enzyme-endosulfan (E-END) complexing for mMDH.