Pseudo-substrates inactivators

A summary of some recently discovered inactivators of E. coli PFL is presented in Table V. Consistent with these compounds acting as mechanism-based or active site-directed inhibitors is the observation of pseudo-first-order inactivation kinetics, substrate protection (by pyruvate or formate for inhibitors that are pyruvate or formate analogs, respectively), and isotope effects on the rates of inactivation by the deuterated analogs. The details of some of these studies, the proposed inactivation mechanisms, and the implications to the normal enzymic reaction are discussed below. 

In direct analogy to the Michaelis-Menten mechanism for reaction of enzyme with a substrate, the inactivator, I, binds to the enzyme to produce an E l complex with a dissociation constant K. A first-order chemical reaction then produces the chemically reactive intermediate with a rate constant k. The activated species may either dissociate from the active site with a rate constant to yield product, P, or covalently modify the enzyme ( 4). The inactivation reaction should therefore be a time-dependent, pseudo-first-order process which displays saturation kinetics. This is verified by measuring the apparent rate constant for the loss of activity at several fixed concentrations of inactivator (Fig. lA). The rate constant for inactivation at infinite [I], itj act (a function of k2, k, and k4), and the Ki can be extracted from a double reciprocal plot of 1/Jfcobs versus 1/ 1 (Fig. IB) (Kitz and Wilson, 1962 Jung and Metcalf, 1975). A positive vertical... 

Behavior of continuous reactors was analyzed in section 5.2 under steady-state operation. This is no longer valid if the enzyme is inactivated during reactor operation. However, if the biocatalyst is reasonably stable (as it should be for an immobilized enzyme to be used continuously), pseudo-steady-state operation can be considered (Vieth et al. 1976). In this way, the initial steady-state (Xi), obtained when the enzyme still remains fully active (E = Eq), can be determined from Eqs. 5.16 and 5.24 for CPBR and CSTR respectively. After certain time of reactor operation (t), the enzyme has suffered inactivation (E = f(t)) and pseudo-steady-state substrate conversion (X) at that time (t) can be estimated from the same Eqs. 5.16 and 5.24. Then,... 

Enzyme Inhibition Assays. The standard Ellman assay (16) was employed in the inhibition studies using electric eel AChE. Most of the kinetic studies were done on confound 1. The results of competitive inhibition assays, carried out by co-incubating 1 with s ibstrate acetylthiocholine at 25""C in phosphate buffer pH=8, showed that the was 80 1M and the K substrate was 144 1M. Preincubation of the enzyme with various concentrations of the inhibitor showed that conpound 1 inactivated the AChE in a time dependent manner, with pseudo first order kinetics below 40 XM. A Kitz-Wilson treatment (17) demonstrated a of 80 JIM and a of 0.35 min" at saturating inhibitor... 

Other unsaturated substrate analogs that have been tried as enzyme inhibitors include allyl amine and allyl alcohol. Allylamine is a pseudo-irreversible inhibitor of flavin-linked monoamine oxidase i.e., in the presence of allylamine, the enzyme shows a time-dependent inactivation that cannot be reversed by dialysis. When radiolabeled allylamine is used, radioactivity is incorporated at the same rate as the enzyme is inhibited. However, inhibition is relieved and radioactivity is removed from the enzyme upon incubation with the substrate, benzylamine. 

A third criterion is that the addition of the real substrate to the reaction linking the affinity label covalently to the protein should diminish the rate of inactivation, which, in the presence of excess labeling reagent, is usually pseudo-first order. 

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