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.