4._PHM Inactivators
There are important differences between an enzyme inhibitor (I) and an enzyme inactivator (IN). An inhibitor forms a reversible enzyme-inhibitor complex: E + I E-I. An inhibitor shows no time-dependence in the percent inhibition at any inhibitor concentration. An inactivator forms an irreversible enzyme-inactivator complex (E-IN)* from the reversible E-IN complex: E + IN E-IN → (E-IN)*. Typically, the (E-IN)* complex results from the formation of a covalent bond between the inactivator and the enzyme. At any concentration of inactivator, the degree of inhibition increases over time as higher concentrations of (E-IN)* accumulate (Morrison & Stone, 1985).
The first report of a PHM inactivator wastrans -4-phenyl-3-butenoate (PBA) (A. F. Bradbury, Mistry, Roos, et al., 1990). In addition to PBA and ring-substituted PBAs (Langella et al., 2010), other PHM inactivators includetrans -styrylthioacetate (Casara et al., 1996), 2-[(phenylethynyl)thio]acetate (Casara et al., 1996), acrylates (Foster, Oldham, & May, 2011; A. G. Katopodis & S. W. May, 1990; Rhodes & Honsinger, 1993), monoethyl fumarate (A. G. Katopodis & S. W. May, 1990), 2-, 3-, and 2,4-alkenoates (Rhodes & Honsinger, 1993), cinnamate and ring-substituted cinnamates (A. F. Bradbury, Mistry, & Smyth, 1990; N.R. McIntyre et al., 2016), N -formyl amides (Klinge, Cheng, Zabriske, & Vederas, 1994), and peptides with a C-terminal vinylglycine (Zabriskie et al., 1994). Treatment of cultured mammalian cells and rats with PBA inhibits PHM activity and the biosynthesis of α-amidated peptides (Abou-Mohamed et al., 2000; Ogonowski et al., 1997). The methyl ester prodrug of PBA was ~10-fold more effective than PBA in inhibiting the growth of tumorigenic rat liver epithelial cells (WB-Ras cells) (Sunman, Foster, Folse, May, & Matesic, 2004).
Most of the PHM inactivators were designed as mechanism-based inactivators to trap the radical intermediate that is likely to form during catalysis (F. Cao & Easton, 2013; Cowley et al., 2016). These are mechanism-based inactivators because O2 and ascorbate are required for inactivation and peptide substrates protect against inactivation. However, the chemistry of inactivation is unclear (F. Cao & Easton, 2013; Driscoll et al., 2000; N.R. McIntyre et al., 2016; Zabriskie et al., 1992). As expected PAL is unaffected by the PHM inactivators (N.R. McIntyre et al., 2016; Moore & May, 1999). Attempts to label PHM using either a 14C-labeled or fluorescently-tagged inactivator have been unsuccessful, with the exception of one early study of PBA-mediated inactivation, in which the labeled protein was not subjected to peptide mapping. Peptide-mapping of cinnamate-inactivated PHM showed no differences compared to the untreated control (N.R. McIntyre et al., 2016). We are unaware of any PAL-specific inactivators.