Bisubstrate analog enzyme inhibitors

Paradoxical at first glance. Recall that phosphonacetyl-l-aspartate (PALA) is a potent inhibitor of ATCase because it mimics the two physiological substrates. However, low concentrations of this unreactive bisubstrate analog increase the reaction velocity. On the addition of PALA, the reaction rate increases until an average of three molecules of PALA are bound per molecule of enzyme. This maximal velocity is 17-fold as great as it is in the absence of PALA. The reaction rate then decreases to nearly zero on the addition of three more molecules of PALA per molecule of enzyme. Why do low concentrations of PALA activate ATCase ... 

While developed as possible therapeutics, bisubstrate analogs have found great utility in the dissection and characterization of enzyme structure and mechanism. As discussed below, bisubstrate analogs have been used extensively in structural studies, where the use of natural substrates would result in catalysis, to investigate the architecture of the active site at the Michaelis complex, and to define structural changes at the active site produced by allosteric effectors. In some cases, bisubstrate analogs that are formed during the reaction (a type of mechanism-based inhibitor) can help to support or eliminate proposed chemical mechanisms. 

In an attempt to avoid problems with the phosphate groups from the CoA moiety, the penultimate intermediate in the synthesis of the bisubstrate inhibitor, A-bromoacetyltryptamine, was tested as a possible affinity label inhibitor or as a substrate for the in situ enzymatic synthesis of the bisubstrate inhibitor through an acylation mechanism. N-Bromoacetyltryptamine did act as an inhibitor of the enzyme, but inhibition could be reversed by dialysis suggesting that the inhibition was not due to a covalent adduct. It was shown that the enzyme catalyzed the acylation of A-bromoacetyltryptamine by CoASH to form compound 6a with a rate enhancement of 3.3 x 10 relative to the uncatalyzed reaction. Ultimately, it was shown that the acylation reaction occurs at the same active site as the acetylation activity. A closer inspection of the kinetics of inhibition by tbe bisubstrate analog 6a resulted in the observation of slow-onset inhibition over the first few minutes of the reaction with a A) value of 84nmol 1 . Owing to its neutrality, A-bromoacetyltryptamine was tested as an inhibitor in vivo. The analog precursor was shown to inhibit melatonin production in norepinephrine-stimulated pinealocytes in a concentration-dependent manner and with low cytotoxicity. 

The use of bisubstrate analog inhibitors in structural experiments of AANAT provided a very powerful tool to probe the subtle differences in the active site leading to either acetyltransferase or acyltransferase activity. Compound 6a was shown to have Aj values of 0.048 and 33 pmoll for the acetyltransferase and acyltransferase activities, respectively. When this compound is cocrystallized with the enzyme, two forms of the inhibitor... 

An extremely important aspect of bisubstrate inhibitors is that they usually utilize multiple binding points to achieve their high affinities for enzymes. The current catechol analog COMT inhibitors rely on the nitro-substituent to provide potency. When uncertainty about the cause of occurrences of hepatoxicity in Parkinson s disease patients on the cocktail therapy was directed at the nitro-substituent, the question quickly arose as to whether the nitro-substituent was required for bisubstrate analog potency. It was shown that utilizing a 4-methyl-phenyl group in place of the nitro-substituent (16) resulted in an inhibitor with only twofold less potency (IC50 value of 2 3 versus 9 nmol When the new inhibitor was modeled into the COMT active site,... 

The first synthetic bisubstrate analog inhibitor 18 was successfully designed for al,2-FucT II by Palcic et al., based on the proposed ion-pair mechanism shown in Scheme 8 [22]. Analog 18, where the Gal unit is attached to the terminal phosfor of GDP through a flexible ethylene linkage, was found to be a competitive inhibitor with respect to both donor and acceptor substrates with K values of 16 and 2.3 pM, respectively. Inhibition studies with the bisubstrate analog also helped establish the kinetic mechanism of the enzyme reaction. These dual competitive inhibition patterns are only consistent with a random kinetic mechanism where either substrate can bind to free enzyme. 

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