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\begin{document}

\title{Peptidylglycine \selectlanguage{greek}α-\selectlanguage{english}amidating monooxygenase as a therapeutic target or
biomarker\selectlanguage{english}}



\author[1]{David Merkler}%
\author[1]{Aidan Hawley}%
\author[2]{Betty Eipper}%
\author[2]{Richard Mains}%
\affil[1]{University of South Florida}%
\affil[2]{UConn Health Center}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
Peptides play a key role in controlling many physiological and
neurobiological pathways. Many bioactive peptides require a C-terminal
\selectlanguage{greek}α-\selectlanguage{english}amide for full activity. The bifunctional enzyme catalyzing
\selectlanguage{greek}α-\selectlanguage{english}amidation, peptidylglycine \selectlanguage{greek}α-\selectlanguage{english}amidating monooxygenase (PAM), is the
sole enzyme responsible for amidated peptide biosynthesis, from
Chlamydomonas reinhardtii to Homo sapiens. Many neuronal and endocrine
functions are dependent upon amidated peptides; additional amidated
peptides are growth promoters in tumors. The amidation reaction occurs
in two steps, glycine \selectlanguage{greek}α-\selectlanguage{english}hydroxylation followed by dealkylation to
generate the \selectlanguage{greek}α-\selectlanguage{english}amide product. Currently, most potentially useful
inhibitors target the first reaction, which is rate-limiting. PAM is a
membrane-bound enzyme that visits the cell surface during peptide
secretion. PAM is then used again in the biosynthetic pathway, meaning
that cell-impermeable inhibitors or inactivators could have therapeutic
value for the treatment of cancer or psychiatric abnormalities. To date,
inhibitor design has not fully exploited the structures and mechanistic
details of PAM.%
\end{abstract}%



\sloppy


\textbf{1. Introduction}

Bioactive peptides play a key role in controlling many complex pathways
including blood glucose (insulin and glucagon, for example), salt and
water balance (vasopressin, atrial natriuretic peptide), appetite
(cholecystokinin, \selectlanguage{greek}α-\selectlanguage{english}melanocyte stimulating hormone, leptin),
reproduction (gonadotropin hormone releasing hormone, follicle
stimulating hormone) and gastrointestinal function (gastrin, motilin).
Each of these peptides is synthesized from a newly synthesized precursor
protein as it moves from its site of synthesis in the endoplasmic
reticulum, through the secretory pathway lumen and into the secretory
granules from which it is released in response to the appropriate
combination of stimuli (Chr\selectlanguage{ngerman}étien \& Mbikay, 2016; Clark \& Lowry, 2016;
Kumar, Mains, \& Eipper, 2016). A limited set of subtilisin-like
endoproteases, carboxypeptidase B-like exoproteases and other
post-translational processing enzymes convert these precursors, which
are often inactive, into active products as they move through the
secretory pathway (\textbf{Fig.1A} ). The activity of many bioactive
peptides requires \selectlanguage{greek}α-\selectlanguage{english}amidation of their C-terminal residue; lacking an
amidated C-terminus, their ability to bind to their receptors (generally
a G Protein Coupled Receptor) is greatly reduced. Consistent with this,
Peptidylglycine \selectlanguage{greek}α-\selectlanguage{english}Amidating Monooxygenase (PAM), the only enzyme known
to catalyze the formation of \selectlanguage{greek}α-\selectlanguage{english}amidated peptides, is an essential
enzyme. Mice lacking PAM survive only until mid-gestation (Czyzyk et
al., 2005) and neither flies nor zebrafish lacking PAM are viable
(Kolhekar, Roberts, et al., 1997; Kumar et al., 2018). Mice having a
single functional \emph{Pam} gene exhibit a wide variety of deficits,
ranging from altered inhibitory synaptic neurotransmission to increased
anxiety-like behavior and an inability to maintain body temperature in a
cold environment (Bousquet-Moore et al., 2010; Gaier, Eipper, \& Mains,
2014; Gaier et al., 2013). Genetic studies have identified \emph{PAM} as
a risk factor for type 2 diabetes (Steinhorsdottir et al., 2014; Thomsen
et al., 2018).

PAM is a bifunctional enzyme: its monooxygenase domain (peptidylglycine
\selectlanguage{greek}α-\selectlanguage{english}hydroxylating monooxygenase, PHM: EC 1.14.17.3) catalyzes the copper-
and ascorbate-dependent \selectlanguage{greek}α-\selectlanguage{english}hydroxylation of its peptidylglycine
substrates; its lyase domain (peptidyl-\selectlanguage{greek}α-\selectlanguage{english}hydroxyglycine \selectlanguage{greek}α-\selectlanguage{english}amidating
lyase, PAL: EC 4.3.2.5; also called peptidylamidoglycolate lyase) then
generates the \selectlanguage{greek}α-\selectlanguage{english}amidated product peptide plus glyoxylate
(\textbf{Fig.1B} ). C-terminal amidation can alter peptide structure,
confer resistance to proteolytic degradation and reduce the effects of
pH on receptor binding (Kahns \& Bundgaard, 1991; Luxmi, Mains, King, \&
Eipper, 2021). Extensive mechanistic studies and structural studies
facilitated the development of PAM inhibitors. In this brief review we
will summarize several successful approaches taken to develop inhibitors
of PHM and discuss observations indicating that PAM may serve as a
useful therapeutic target or biomarker.

\selectlanguage{greek}\textbf{2. Peptide α-Amidation in Two Steps}\selectlanguage{english}

\textbf{2A. The PHM Reaction:} Progress in studies on the formation of
\selectlanguage{greek}α-\selectlanguage{english}amidated peptides depended crucially on three key findings. First,
studies conducted from the 1950's to the early 1980's made it clear that
the proprotein precursors to peptide-amides always had a Gly residue
located to the C-terminal side of the residue that was to be \selectlanguage{greek}α-\selectlanguage{english}amidated
(reviewed in (Kumar, Mains, et al., 2016)) (\textbf{Fig.1A}
and\textbf{B} ). Second, development of the first useable enzymatic
assay of peptide amidation by Bradbury, Finnie and Smyth (A.F. Bradbury,
Finnie, \& Smyth, 1982). Third, the realization that pituitary cells
maintained in serum-free culture medium efficiently stored and secreted
POMC products, but were unable to produce \selectlanguage{greek}α-\selectlanguage{english}amidated peptides (Eipper,
Glembotski, \& Mains, 1983). Taken together, these observations lead to
the conclusion that the reaction depended on copper ions, reduced
ascorbic acid and oxygen, followed by the purification of PHM, PAL and
PAM from a variety of sources (Eipper et al., 1991; Murthy, Keutmann, \&
Eipper, 1987; Murthy, Mains, \& Eipper, 1986; P. P. Tamburini, Young,
Jones, Palmesino, \& Consalvo, 1990) and the cloning and expression of
PAM cDNAs from several species (Bertelsen et al., 1990; Eipper et al.,
1987; Glauder, Ragg, Rauch, \& Engels, 1990; Mizuno et al., 1987; Ouafik
et al., 1992; Stoffers, Green, \& Eipper, 1989; Stoffers, Ouafik, \&
Eipper, 1991). Although the lyase reaction catalyzed by PAL can occur at
elevated pH in the absence of enzyme, phylogenetic studies support the
presence of a bifunctional, integral membrane PAM protein
(\textbf{Fig.1C} ) in the last eukaryotic common ancestor (B\selectlanguage{ngerman}äck, Mains,
\& Eipper, 2021; Kumar, Mains, et al., 2016).

Most of the enzymological and all of the crystallographic studies of PHM
and PAL have been carried out using the soluble, protease-resistant
catalytic cores of PHM (PHMcc) and PAL (PALcc) (\textbf{Fig.1C} ).
Endoproteolytic digestion of lysates prepared from mammalian cells
expressing exogenous PAM and of tissues that express high levels of
endogenous PAM revealed that both catalytic activities were remarkably
resistant to proteolytic degradation (Husten \& Eipper, 1994; Husten,
Tausk, Keutmann, \& Eipper, 1993). For rat PAM, PHMcc consists of
residues 42--356 (Eipper, Quon, Mains, Boswell, \& Blackburn, 1995;
Kolhekar, Keutmann, Mains, Quon, \& Eipper, 1997) and PALcc consists of
residues 498--820 (Kolhekar et al., 2002; Kolhekar, Quon, Berard, Mains,
\& Eipper, 1998). The crystal structures of recombinant PHMcc and PALcc
have been extensively studied (Chufan, De, Eipper, Mains, \& Amzel,
2009; Prigge, Eipper, Mains, \& Amzel, 2004; Prigge, Kolhekar, Eipper,
Mains, \& Amzel, 1997; Siebert et al., 2005). PAM, PHM, and PAL purified
from natural sources and soluble, recombinant PHM, PAL and bifunctional
PAM have provided additional insight into the reaction mechanism
(\textbf{Fig.1C} ) (Bertelsen et al., 1990; Miller et al., 1992). To
date, it has not been possible to obtain high resolution structural data
for any bifunctional PAM proteins. Studies of the PHM and PAL components
of bifunctional PAM are accomplished by employing the appropriate
assays: O\textsubscript{2} consumption from a glycine-extended peptide
is specific for PHM and glyoxylate production from a carbinolamide is
specific for PAL. Although few studies have utilized human PAM, PHM or
PAL, the highly conserved nature of the PHM and PAL active sites (Bäck
et al., 2021; Kumar, Mains, et al., 2016) suggests that the catalytic
properties of all of the various PAM, PHM and PAL proteins mentioned
above are quite similar. Readers are encouraged to review the cited
references if the exact source of the proteins is important to their
research goals. Despite many attempts to express fully active PAM in
bacterial, yeast, and insect systems, mammalian cell lines remain the
system of choice. PHMcc has been expressed in \emph{E. coli} with an
N-terminal fusion to thioredoxin, but with a yield of only
\textasciitilde{}100 \selectlanguage{greek}µ\selectlanguage{english}g/liter of \emph{E. coli} (Handa, Spradling,
Dempsey, \& Merkler, 2012). Production of recombinant rat PHMcc, PALcc
and rat MTC PAM in Chinese hamster ovary (CHO) cells has yielded
\textgreater{}100 mg of pure enzyme (Bauman, Ralle, \& Blackburn, 2007;
Miller et al., 1992), sufficient enzyme to support spectroscopic studies
(Blackburn, Rhames, Ralle, \& Jaron, 2000; Eipper et al., 1995; Evans,
Blackburn, \& Klinman, 2006; Jaron \& Blackburn, 1999), structural
biology (Chufan et al., 2009; Prigge et al., 1997; Prigge, Kolhekar,
Eipper, Mains, \& Amzel, 1999), and amidated peptide production at the
multi-gram level (Ray et al., 1993).

PHM function requires two bound copper ions, Cu\textsubscript{H} (with
three His ligands) and Cu\textsubscript{M} (with two His and one Met
ligand) (Maheshwari et al., 2018; Prigge et al., 2004; Prigge et al.,
1999; Siebert et al., 2005). Copper is easily lost from PHM, meaning
that assaying PHM activity in crude lysates or in serum samples (Gaier
et al., 2012) requires the addition of exogenous copper and analysis of
purified PHM and PAM often requires replacement of copper lost during
purification (Blackburn et al., 2000; Evans et al., 2006; Jaron \&
Blackburn, 1999; Murthy et al., 1986). The first step in the amidation
reaction is the reduction of Cu(II)\textsubscript{H} and
Cu(II)\textsubscript{M} to Cu(I) by ascorbate (\textbf{Fig.2A} , Step
1). Studies show that the kinetic mechanism is equilibrium ordered, with
the glycine-extended substrate binding first to reduced enzyme
(\textbf{Fig. 2A} , Step 2), followed by the binding of
O\textsubscript{2} to Cu(I)\textsubscript{M} (\textbf{Fig.2A} , Step 3).
Formation of the PHM product, the (\emph{S} )-\selectlanguage{greek}α-\selectlanguage{english}hydroxyglycine
derivative (Cowley, Tian, \& Solomon, 2016; Ping, Mounier, \& May,
1995), proceeds from the
PHM-Cu(I)\textsubscript{H}-Cu(I)\textsubscript{M}-substrate-O\textsubscript{2}complex
(\textbf{Fig. 2A} , Step 4) and regenerates oxidized
PHM-Cu(II)\textsubscript{H}-Cu(II)\textsubscript{M}, preparing the
enzyme for the next catalytic cycle (\textbf{Fig.2A} , Step 5).

The chemical mechanism for PHM catalysis has been intensively debated
(Chen, Bell, Eipper, \& Solomon, 2004; Cowley et al., 2016; W.A.
Francisco, Merkler, Blackburn, \& Klinman, 1998; Jaron \& Blackburn,
1999; Klinman, 2008; Kulathila, Merkler, \& Merkler, 1999; Ping et al.,
1995); it is agreed that a substrate-based radical on the \selectlanguage{greek}α-\selectlanguage{english}carbon of
the glycine is an intermediate and that a copper-superoxo species,
Cu\textsubscript{M}(II)-O\textsubscript{2}\textsuperscript{*}, is
responsible for hydrogen atom abstraction from the substrate glycine.
Recent work from Wu et al. (Wu et al., 2019) suggests that an unusual
(\selectlanguage{greek}\emph{μ} -\selectlanguage{english}O\textsuperscript{*})(\selectlanguage{greek}\emph{μ}
-\selectlanguage{english}OH)Cu(II)\textsubscript{M}Cu(II)\textsubscript{H}is the species
responsible for hydrogen atom abstraction from substrate. In addition to
the uncertainties about the reduced oxygen species responsible for
hydrogen atom abstraction, there remain questions about the structure of
the bifunctional PAM, binding site(s) for ascorbate, the electron
transfer pathways between the two copper atoms and from the copper atoms
to the substrate during catalysis, potential domain motion in PHM to
reduce the 11\selectlanguage{ngerman}Å distance between the Cu(II), and the possibility of
carbinolamide channeling between the PHM and PAL active sites in PAM.
Note that development of the current cohort of PHM inhibitors (discussed
below) has progressed without definitive answers to these questions.
Thus, the PHM and PAL inhibitors developed to date are substrate
analogs. The rational design of second and third generation PHM
inhibitors would benefit from a resolution of the mechanistic
uncertainties, fostering the design of tight-binding transition-state
analog inhibitors.

\textbf{2B. The PAL Reaction:} Like PHM, PAL is a metalloenzyme; PAL
contains a bound Zn(II) and a bound Ca(II) (Bell et al., 1997; Chufan et
al., 2009; De, Bell, Blackburn, Mains, \& Eipper, 2006). PAL catalyzes
the dealkylation of the (\emph{S} )-\selectlanguage{greek}α-\selectlanguage{english}hydroxyglycine derivative to the
corresponding amide and glyoxylate (\textbf{Fig.2A} , Step 5)
(Katopodis, Ping, \& May, 1990; Perkins, Husten, \& Eipper, 1990). To
date, evidence about channeling of the peptidyl-\selectlanguage{greek}α-\selectlanguage{english}hydroxyglycine product
from PHM to PAL is conflicting (W.A. Francisco et al., 1998; Moore \&
May, 1999). One major site for PHM and PAL catalysis is the acidic
secretory granules with an internal pH\textasciitilde{}5 (Mains \& May,
1988). The \selectlanguage{greek}α-\selectlanguage{english}hydroxyglycine derivative (a carbinolamide) is stable at
this pH (Bundgaard \& Kahns, 1991); thus, PAL is required for effective
amide formation. The bound Ca(II) has a structural role in PAL, while
the bound Zn(II) is likely involved in catalysis (Chufan et al., 2009;
Takahashi et al., 2009). The precise role played by Zn(II) is not yet
clear; suggestions include Zn(II)-bound water or hydroxide serving as a
general base for proton abstraction from the hydroxyl group (Bell et
al., 1997; Takahashi et al., 2009) and Zn(II)-coordination to the
hydroxyl group to lower its pK\textsubscript{a}, facilitating proton
transfer to a tyrosyl phenolate in the active site (Chufan et al.,
2009). Model studies by Tenn \emph{et al} . (Tenn et al., 2007) suggest
that carbinolamide dealkylation is not base-catalyzed and is independent
of the pK\textsubscript{a} of the hydroxyl group of the carbinolamide.
Instead, these authors argue that carbinolamide dealkylation is
dependent upon the nucleofugality of the departing amidate. If so, the
PAL-bound Zn(II) could assist in the departure of the amidated product.
Additional studies of PAL catalysis are required to address these
mechanistic questions.

In the few cases examined, tissue lysates contained substantially more
\selectlanguage{greek}α-\selectlanguage{english}amidated peptide than peptidylglycine precursor and levels of
peptidyl-\selectlanguage{greek}α-\selectlanguage{english}hydroxyglycine intermediate were well below peptidylglycine
levels (Yin et al., 2011). These observations are consistent with the
higher V\textsubscript{max} values reported for PAL vs PHM. As discussed
below, the PAL domain plays an essential role in the ability of cells to
retrieve membrane PAM from the cell surface. This suggests that
inhibitors targeted to the active site of PAL might provide an effective
means of inhibiting peptide amidation by reducing the amount of PAM
available to amidate newly synthesized peptidylglycine substrates
entering immature secretory granules. It is intriguing that the PAM loci
most frequently associated with human diseases are in the PAL domain,
not the PHM domain (Steinhorsdottir et al., 2014; Thomsen et al., 2018).

\textbf{3.Effective PHM and PAL Inhibitors Were Developed} .

For a period of about 25 years, from 1990 to 2015, there was
considerable interest in developing PHM inhibitors and inactivators (Ali
et al., 2015; Bolkenius, Ganzhorn, Chanal, \& Danzin, 1997; F. Cao et
al., 2011; A.G. Katopodis \& S.W. May, 1990; Langella et al.,
2010)\textbf{.} The hope was that a PHM-targeted compound would be
therapeutically useful in the treatment of human disease and/or would
contribute to our understanding of PHM. Compounds that would intercept
the radical intermediate were developed (Zabriskie, Cheng, \& Vederas,
1992; Zabriskie, Klinge, Szymanski, Cheng, \& Vederas, 1994) and PAM
labeling with mechanism-based inactivators (suicide substrates) was used
to try to identify the active site amino acids critical to catalysis.
There was less interest in developing PAL-targeted compounds, the
thought being that the non-enzymatic dealkylation of the accumulated
carbinolamide intermediate would supply sufficient levels of the
amidated product to alleviate PAL inhibition; as discussed above,
PAL-targeted compounds that altered the trafficking of PAM could still
prove useful in manipulating amidation \emph{in vivo} .

Many drugs exhibit cell toxicity. Such therapeutics have greater
toxicity towards diseased cells relative to healthy cells and must be
used properly in treating a disease. In addition, ongoing research will
lead to ``molecular zip codes'', a protein or a small molecule that
binds tightly and specifically to uniquely target diseased cells (Enb\selectlanguage{ngerman}äck
\& Laakkonen, 2007). The attachment of a drug to a molecular zip code
would enable the delivery of the drug specifically to the diseased cells
-- a strategy enabling the safe use of highly toxic drugs to treat human
disease. A toxic PHM-specific inhibitor could be valuable
therapeutically if appended to the appropriate molecular zip code. The
production of amidated autocrine growth factors by tumor cells suggests
that high-affinity cell-impermeant PHM inhibitors might prove useful in
controlling the growth of these cells. Most neurons and endocrine cells
cleave PHM from PAL within secretory granules, and release the PHM along
with peptide. These cells would presumably ingest less of the toxic
PHM-specific inhibitor than would cancerous cells, since tumor cells
usually leave much of their PAM intact while making the amidated
autocrine growth factors which enhance tumorigenicity.

Another use of biomedical significance for a PHM-targeted compound would
be as a PAM-specific imaging agent. PHM is a biomarker for specific
cancers (Jiménez et al., 2003; Rocchi et al., 2004; Scopsi et al., 1998;
Thouënnon et al., 2007), post-polio syndrome (Gonzalez et al., 2009),
and neural dysfunction (Bousquet-Moore et al., 2010). In sum, the
toxicity concern about a PHM inhibitor/inactivator is short-sighted and
is limiting work to develop PHM-dependent imaging agents and provides
few options when a PHM-specific molecular zip code becomes available.

\textbf{3A. PHM Inhibitors:} The inhibitory effects of divalent metal
ion chelators on peptide amidation became apparent as soon as different
buffers were tested for use in enzyme assays of tissue lysates (Eipper,
Mains, \& Glembotski, 1983). Inhibition by diethyldithiocarbamate (DDC)
was reversed only by the addition of Cu(II), providing the first
evidence that PAM/PHM catalysis was copper-dependent; no other divalent
metal, including Zn(II), restored activity (Eipper, Mains, et al., 1983;
Eipper, Park, Keutmann, \& Mains, 1986). Other metal chelators like EDTA
(Merkler, Kulathila, Young, Freeman, \& Villafranca, 1993) and
disulfiram inhibit PAM/PHM reversibly (Mains, Park, \& Eipper, 1986).
Disulfiram (Antabuse) is used for the treatment of alcohol abuse
(Kranzler \& Soyka, 2018) and has anti-cancer activity (Corsello et al.,
2020). The possible link between the anti-cancer activity and PHM
inhibition has not been directly investigated. Disulfiram has been used
to inhibit \selectlanguage{greek}α-\selectlanguage{english}amidated peptide production in cultured mammalian cells and
in rats (Mains et al., 1986; Mueller \& Altarac, 1995; Rondeel et al.,
1995).

Most of the PHM inhibitors developed to date likely bind to the peptide
substrate site within the PHM active site (Prigge et al., 1997). A
review of the substrates oxidized by PHM is required to provide context
for the PHM inhibitors. Only peptides with a C-terminal glycine are PHM
substrates \emph{in vivo} (K.A. Johnson, Paisley-Flango, Tangarone,
Porter, \& Rouse, 2007). Synthetic peptides with a C-terminal D-Ala are
substrates leading to the corresponding \selectlanguage{greek}α-\selectlanguage{english}amidated peptide and pyruvate
(Landymore-Lim, Bradbury, \& Smyth, 1983). However, the D-Ala-extended
peptides are relatively poor substrates with low
V\textsubscript{max}/K\textsubscript{m} ratios. The
V\textsubscript{max}/K\textsubscript{m} ratio provides an estimate of
the first-order rate constant for the conversion of substrate to product
and is obtained using a substrate concentration far below its
K\textsubscript{m} value; when comparing different substrates, it
provides a measure of enzyme specificity (K. A. Johnson, 2019). For
example, the V\textsubscript{max}/K\textsubscript{m} ratio for\emph{N}
-dansyl-Tyr-Val-D-Ala is 0.06 times the ratio for N-dansyl-Tyr-Val-Gly;
the V\textsubscript{max}/K\textsubscript{m} ratio for \emph{N}
-benzoyl-D-Ala is 0.0005 times the ratio for\emph{N} -benzoylglycine
(Merkler et al., 2008). Systematic studies to determine the effect of
the penultimate amino acid on the kinetics of amidation showed that
glycine-extended peptides with a penultimate aromatic, hydrophobic, or
sulfur-containing amino acid had the highest
V\textsubscript{max}/K\textsubscript{m} ratios while those with a
penultimate Lys or Arg had the lowest
V\textsubscript{max}/K\textsubscript{m} ratios (Merkler et al., 1993; P.
P. Tamburini et al., 1990). \selectlanguage{greek}α-\selectlanguage{english}Amidated peptides with a C-terminal amino
acid amide for all 20 of the common amino acids are known (Eipper,
Stoffers, \& Mains, 1992). Studies aimed at producing amidated peptides
in cell lines using endogenous or exogenous PAM indicate that the
general rules for effective amidation developed by Tamburini et al. are
not directly applicable to the conditions encountered as PAM and its
substrates move through the secretory pathway in cells (Chew et al.,
2005). A free carboxylate is required on the C-terminal glycine for
amidation. The methyl or ethyl esters are not substrates, but are
inhibitors of relatively low affinity (P.P. Tamburini et al., 1988).
Similarly, the amidated products are low affinity inhibitors
(Glembotski, Eipper, \& Mains, 1984).

\selectlanguage{greek}α-\selectlanguage{english}Amidated peptide hormones range considerably in length, from 3 amino
acids for TRH to 52 amino acids for adrenomedullin. Much larger proteins
that terminate with a C-terminal amide and generate \selectlanguage{greek}α-\selectlanguage{english}amidated
chemomodulatory peptides have been identified in the green
algae,\emph{Chlamydomonas reinhardtii} (Luxmi, Kumar, Mains, King, \&
Eipper, 2019). When expressed exogenously, for therapeutic purposes,
monoclonal antibodies whose heavy chains terminate with a glycine are
often \selectlanguage{greek}α-\selectlanguage{english}amidated (Skulj, Pezdirec, Gaser, Kreft, \& Zorec, 2014);
elimination of the low levels of PAM expressed in the mammalian cells
used to produce these monoclonal antibodies eliminates their \selectlanguage{greek}α-\selectlanguage{english}amidation
(Skulj et al., 2014). Dipeptides, X-Gly, are poor PHM substrates,
exhibiting low V\textsubscript{max}/K\textsubscript{m} ratios (Wilcox et
al., 1999) and, in general, longer peptide substrates exhibit higher
V\textsubscript{max}/K\textsubscript{m} ratios (Chew et al., 2005).
Modeling of \emph{N} -\selectlanguage{greek}α-\selectlanguage{english}acetyl-3,5-diiodotyrosylglycine into the
structure of PHMcc indicates that the carboxylate of the glycyl residue
forms a salt-bridge with Arg\textsuperscript{240} (Prigge, Mains,
Eipper, \& Amzel, 2000). This arginine is highly conserved across PHM
sequences and likely provides a rationale for the importance of the free
carboxylate for substrate binding. PHMcc is comprised of two domains of
approximately the same size that are connected by a single strand; the
single copper atom bound to each domain is poised at the edge facing the
solvent-filled cleft separating the domains (Prigge et al., 1997). The
interiors of both domains are hydrophobic and the copper atoms are
separated by 11\selectlanguage{ngerman}Å (Prigge et al., 1997). Upon reduction, there is
virtually no change in PHMcc structure (Prigge et al., 2004). The large,
hydrophobic interdomain region of PHMcc is necessary for the amidation
of relatively large peptide substrates and is consistent with a
preference for penultimate hydrophobic amino acids in the substrate. How
electron transfer and radical stability are accomplished in the large,
solvent accessible interdomain region of PHM are amongst the unanswered
questions about PHM catalysis. A PHM structure with a large peptide
substrate bound might provide insight about these issues.

Strikingly, PHM and PAL will accept a wide variety of non-peptide
substrates possessing a moiety that is equivalent to the C-terminal
glycine, including the \emph{N} -acylglycines, hippurate (\emph{N}
-benzoylglycine) and substituted hippurates (Merkler et al., 2008), and
the bile acid glycine conjugates (King et al., 2000). A carbinolamide
intermediate is known to form as these substrates are converted into the
amide and glyoxylate. Consistent with the preference for a hydrophobic
penultimate amino acid for peptide substrates, the
V\textsubscript{max}/K\textsubscript{m} ratio for \emph{N} -acylglycine
amidation increases as acyl-chain length increases. Early studies by
Katopodis and May demonstrated that PHM could also catalyze
sulfoxidation, amine \emph{N} -dealkylation, and \emph{O} -dealkylation
(A.G. Katopodis \& S.W. May, 1990) (\textbf{Fig. 2B, C, D} ). The PHM
catalyzed \emph{O} -dealkylation of the imino-oxy acetates yields the
corresponding oximes and glyoxylate in a PAL-independent manner (N.R.
McIntyre, Lowe, Battistini, Leahy, \& Merkler, 2016; N. R. McIntyre,
Lowe, \& Merkler, 2009; Schade, Kotthaus, Hungeling, Kotthaus, \&
Clement, 2009). Scorpions, which rely on a variety of amidated peptides
to attack the ion channels of their prey, have retained separate genes
encoding integral membrane PAM, soluble PHM and soluble PAL
(Delgado-Prudencio, Possani, Becerril, \& Ortiz, 2019), leading the
authors to speculate that the PHM secreted in scorpion venom might
catalyze some of these additional reactions. A complete understanding of
PHM catalysis must account for the alternative reactions catalyzed by
this enzyme.

A series of N-blocked Phe-(D,L)-homocysteine peptide analogs were
synthesized and evaluated as PAM inhibitors (Erion, Tan, Wong, \& Jeng,
1994; Jeng, Fujimoto, Chou, Tan, \& Erion, 1997) (\textbf{Fig. 3A} ).
Members of this series with IC\textsubscript{50} values of 8-15 nM
possess an \emph{N} -hydrocinnamoyl, \emph{N} -2-naphthoyl, or\emph{N}
-2-indolyl moiety conjugated to the Phe residue. Synthesis of the benzyl
ester of\emph{N} -2-naphthoyl-L-phenylalanyl-(D,L)-homocysteine as a
prodrug increased the IC\textsubscript{50} from 10 to 8,000 nM, but 10
\selectlanguage{greek}µ\selectlanguage{english}M free carboxylate or benzyl ester showed approximately 50\% inhibition
of PHM in cultured rat dorsal root ganglion neurons, (Jeng et al.,
1997). The potency of this series of compounds mirrors the substrate
specificity information from the glycine-extended peptides. The partial
PHM inhibition observed in the dorsal root ganglion neurons identifies
cell penetration as a challenge for the future clinical use of these
compounds. The disappointing results observed using dorsal root ganglion
neurons for the benzyl ester suggest that the benzyl ester is not the
optimal prodrug, at least, for this series of PHM inhibitors. The
tight-binding of the homocysteine peptide analogs likely results from
the coordination of sulfur atom to Cu\textsubscript{M} (\textbf{Fig. 3B}
). The binding of thiorphan (K\textsubscript{M} = 82 \selectlanguage{greek}µ\selectlanguage{english}M), tiopronin
(K\textsubscript{M} = 33 \(u\)M), and captopril
(K\textsubscript{i}\textasciitilde{}100 \selectlanguage{greek}µ\selectlanguage{english}M), all compounds with a free
sulfhydryl group, was attributed to coordination of the sulfur atom to
one of PHM-bound Cu(II) atoms (N. R. McIntyre, Lowe, Chew, Owen, \&
Merkler, 2006; Mueller, Driscoll, \& Eipper, 1999).

One class of compounds investigated as PHM inhibitors are the
glycolates, R-CO-O-CH\textsubscript{2}-COOH; the R-groups described
include an acyl group (short- and long-chain), a benzoyl group, a
phenylacetyl-group, an \emph{N} -acetyl-amino acid, and\emph{N}
-acetyl-peptides (Barratt et al., 2004; F. Cao et al., 2011; A.G.
Katopodis \& S.W. May, 1990; Morris et al., 2012; Ping et al., 1995)
(\textbf{Fig. 3A} ). The glycolates are analogs of the glycine-extended
substrates and were clear substrate analogs for consideration as
inhibitors. The glycolates are PHM-specific and have no effect on PAL
(Moore \& May, 1999). We have summarized the data for the glycolate and
structurally similar inhibitors in \textbf{Table 1A} and, from these
data, we note the following trends. A number of the glycolate inhibitors
exhibit K\textsubscript{i} or IC\textsubscript{50} values \textless{}5
\selectlanguage{greek}µ\selectlanguage{english}M, meaning that this class of compounds represent a good starting point
for the future development of higher affinity PHM inhibitors. While the
C-terminal glycolate analogs of two \selectlanguage{greek}α-\selectlanguage{english}amidated peptide precursors
(oxytocin and calcitonin) exhibit IC\textsubscript{50} values of 2-12
\selectlanguage{greek}µ\selectlanguage{english}M, the structurally simpler\emph{O} -acylglycolates, like \emph{O}
-lauroylglycolate
(CH\textsubscript{3}-(CH\textsubscript{2})\textsubscript{10}-CO-O-CH\textsubscript{2}-COOH),
exhibit sub-\selectlanguage{greek}µ\selectlanguage{english}M affinities, against PHM proteins from cultured mammalian
cells. Thus, a future tight-binding glycolate inhibitor might be
possible without the complications associated with peptide synthesis.
Another encouraging observation is the differences in affinity between
PHM proteins for some of the glycolate inhibitors. For example,\emph{O}
-lauroylglycolate exhibits an IC\textsubscript{50} of 60 nM for human
lung cancer PHM and an IC\textsubscript{50} of 35 \selectlanguage{greek}µ\selectlanguage{english}M for frog PHM, a
ratio of IC\textsubscript{50} values of \textasciitilde{}600. This
suggests that tissue-, organism-, or disease-state-specific PHM
inhibitors may be possible. The stereochemistry of the penultimate group
has a dramatic effect on the affinity of the glycolate inhibitors. The
ratio of the K\textsubscript{i} values for the D-amino acid
glycolate/L-amino acid glycolate is 40-50 for the\emph{N}
-acetyl-D-Phe-O-CH\textsubscript{2}-COOH/\emph{N}
-acetyl-L-Phe-O-CH\textsubscript{2}-COOH pair and for the\emph{N}
-acetyl-D-Leu-O-CH\textsubscript{2}-COOH/\emph{N}
-acetyl-L-Leu-O-CH\textsubscript{2}-COOH pair. An even stronger
preference for an L-amino acid at the penultimate position was
identified for the glycine-extended peptide substrates;\emph{N}
-Acetyl-L-Phe-Gly binds to PHM [?]700-fold more tightly than\emph{N}
-acetyl-D-Phe-Gly (Ping et al., 1995). Future tight-binding glycolate
inhibitors could take advantage of the stereochemical preference for the
penultimate position.

Cao et al. (F. Cao et al., 2011) synthesized a series of thioglycolates,
R-CO-S-CH\textsubscript{2}-COOH, to determine any effect of the sulfur
atom on the affinity of the PHM inhibitor (\textbf{Fig. 3A}
and\textbf{Table 1B} ). In general, the thioglycolates inhibit with
higher affinity, with a decrease in the IC\textsubscript{50} value by a
factor of 2-25 relative to the corresponding glycolate. The greatest
difference was observed for the\emph{N}
-acetyl-Phe-O-CH\textsubscript{2}-COOH/\emph{N}
-acetyl-Phe-S-CH\textsubscript{2}-COOH pair, with a ratio of
IC\textsubscript{50} values \textgreater{} 80 for frog PHM. Again, these
results suggest that sufficient differences exist between PHM proteins
to enable the design of an inhibitor targeted against a specific PHM.

PHM inhibition by the glycolates and the thioglycolates coupled with the
discovery of \emph{N} -acylglycines as PHM substrates lead to a
structurally related set of PHM inhibitors, the\emph{S}
-(thiocarbonyl)thioglycolates (R--CS--S--CH\textsubscript{2}--COOH)
(\textbf{Fig. 3A} and\textbf{Table 1C} ). \emph{S}
-(Thiolauroyl)thioglycolate,
CH\textsubscript{3}-(CH\textsubscript{2})\textsubscript{10}-CS-S-CH\textsubscript{2}-COOH,
is the tightest binding inhibitor within this set of compounds, with a
K\textsubscript{i} = 540 nM, a relatively high affinity for a
ground-state, substrate analog. A direct comparison between structurally
related glycolates, thioglycolates, and the\emph{S}
-(thiocarbonyl)thioglycolates to define whether PHM binds most tightly
to either the -CS-S- group, the -CO-S- group, or the -CO-O- group is not
possible because the PHM protein used each study was different. For
example, the differences in K\textsubscript{i} or IC\textsubscript{50}
values for
C\textsubscript{6}H\textsubscript{5}-CH\textsubscript{2}-CS-S-CH\textsubscript{2}-COOH
(8 \selectlanguage{greek}µ\selectlanguage{english}M for rat MTC PHM),
C\textsubscript{6}H\textsubscript{5}-CH\textsubscript{2}-CO-S-CH\textsubscript{2}-COOH,
(20 \selectlanguage{greek}µ\selectlanguage{english}M for frog PHM), and
C\textsubscript{6}H\textsubscript{5}-CH\textsubscript{2}-CO-O-CH\textsubscript{2}-COOH
(2 \selectlanguage{greek}µ\selectlanguage{english}M for human H889 cell PHM) might result from the different sources
of PHM. The \emph{S} -(thiocarbonyl)thioglycolate study does provide a
direct comparison of K\textsubscript{i} values between rat MTC PHM and
German cockroach PHM for six of these inhibitors; differences were
found, with
C\textsubscript{6}H\textsubscript{5}-CH\textsubscript{2}-CS-S-CH\textsubscript{2}-COOH
binding to cockroach PHM with \textasciitilde{}10-fold higher affinity
(\textbf{Table 1} ). Again, these data point towards the future
development of inhibitors targeting PHM in a species-specific manner.

In their focus on the synthesis of PHM inactivators designed to capture
the substrate-based radical that likely forms during catalysis, the
Vederas group identified new PHM inhibitors. Included in this group are
D-Phe-L-Phe-\selectlanguage{greek}α-\selectlanguage{english}cyclopropylglycine (Andrews, O'Callaghan, \& Vederas,
1997) and tripeptides with a C-terminal D- or L-styrylglycine (Zabriskie
et al., 1992) (\textbf{Fig. 3A} ). PHM is inactivated by\emph{trans}
-4-phenyl-3-buteonate (\emph{trans} -styrylacetate) (A. F. Bradbury,
Mistry, Roos, \& Smyth, 1990). The cyclopropylglycine-containing
tripeptide does not inactivate PHM and binds with low affinity,
K\textsubscript{i} \textgreater{} 5 mM, additional evidence that PHM
does not readily accommodate any substitution on the glycyl \selectlanguage{greek}α-\selectlanguage{english}carbon.
None of the styrylglycine-containing peptides inactivate PHM, but all
inhibit with IC\textsubscript{50} values of 100 to 450 \selectlanguage{greek}µ\selectlanguage{english}M. This
indicates that the styryl moiety must be optimally positioned in the PHM
active site for inactivation to occur. Even a small repositioning of the
styryl group eliminates its ability to inactivate PHM; neither \emph{N}
-acetyl-D- nor\emph{N} -acetyl-L-styrylglycine inactivates PHM. Little
difference in the IC\textsubscript{50} values of the L- and
D-styrylglycine containing inhibitors was found (Zabriskie et al.,
1992). Thus, the importance of the stereochemistry at the penultimate
position for PHM affinity found for the glycine-extended substrates and
the glycolate inhibitors (Ping et al., 1995) was not observed for the
styrylglycine-containing inhibitors. The significance of these
differences is currently unknown and could be resolved by \emph{in
silico} modeling or crystallographic analysis of appropriate PHM-ligand
complexes. The information gained from such studies could prove useful
for the future design of tight-binding PHM inhibitors.

One clear outcome from the work carried out on PHM inhibitors is
preference for hydrophobicity in groups attached to the glycine or
glycine analog. Consistent with this observation are reports of
hydrophobic organic acids that inhibit PAM with relatively low affinity
Examples from this group of PHM inhibitors include
1-(carboxymethyl)-3,5-diphenyl-2-methylbenzene (IC\textsubscript{50} =
550 \selectlanguage{greek}µ\selectlanguage{english}M) (Cutler et al., 1998), urocanic acid (K\textsubscript{I} = 10
mM) (Merkler et al., 2008), and 4-pentenoic acid (IC\textsubscript{50} =
60 mM) (Rhodes \& Honsinger, 1993).

One other PHM inhibitor, mimosine (\textbf{Fig.3A} ), differs from the
rest. Mimosine is a toxic, heterocyclic, non-protein amino acid produced
by plants (Nguyen \& Tawata, 2016), which is known to inhibit the
mechanistically-related enzyme, dopamine \selectlanguage{greek}β-\selectlanguage{english}monooxygenase (Hashiguchi \&
Takahashi, 1977). Mimosine is competitive vs. ascorbate, determined by
varying the concentrations of mimosine and ascorbate at one-fixed
concentration of glycine-extended substrate, yielding a
K\textsubscript{I} of 4.0 \selectlanguage{greek}µ\selectlanguage{english}M for mimosine (Miller et al., 1992).\emph{In
silico} modeling reveals that mimosine binds to PHM between the two
copper atoms, forming hydrogen bonds to the backbone carbonyls of
Pro-268 and Leu-270 and to the side chain amide of Gln-269
(\textbf{Fig.3A} ). Additional \emph{in silico} modeling suggests that
mimosine and ascorbate bind at approximately the same site in PHM
(\textbf{Fig.3B} ), consistent with mimosine being competitive vs.
ascorbate. The appropriate attachment of mimosine (or a mimosine analog)
to an inhibitor that binds to the peptide site in PHM could yield a
bifunctional inhibitor of high potency (\textbf{Fig.3C} ).

\textbf{3B. PAL inhibitors:} The metals bound to PAL remain bound during
its purification. Like PHM, metal chelators can inhibit PAL by removing
the bound Zn(II). Activity is restored to inactive apo-PAL by the
addition of Zn(II) and other divalent metal ions (Bell et al., 1997).
Unlike PHM, PAL does not exhibit stereochemical preference at the
penultimate position; the kinetic constants for\emph{N}
-acetyl-L-Phe-\selectlanguage{greek}α-\selectlanguage{english}hydroxyglycine and\emph{N}
-acetyl-D-Phe-\selectlanguage{greek}α-\selectlanguage{english}hydroxyglycine are very similar (Ping et al., 1995).
C-Terminal pyruvate-extended amino acids,
R-CO-CH\textsubscript{2}-CO-COOH, are inhibitors of PAL with
K\textsubscript{i} values \textgreater{}15 \selectlanguage{greek}µ\selectlanguage{english}M.\emph{N}
-Acetyl-L-Phe-pyruvate is the best PAL inhibitor reported, with a
K\textsubscript{i} of 0.24 \selectlanguage{greek}µ\selectlanguage{english}M. The pyruvate-extended amino acids weakly
inhibit PHM, being competitive with ascorbate and a
\textasciitilde{}100-fold lower potency (Mounier et al., 1997). The
related compound,
C\textsubscript{6}H\textsubscript{5}-CH=CH-SO-CH\textsubscript{2}-COOH
(\emph{trans} isomer), shows no inhibition or inactivation of PHM at 3
mM, but was not evaluated as a PAL inhibitor (Casara, Banzhorn,
Philippo, Chanal, \& Danzin, 1996). We are not aware of any other work
to develop a PAL-specific inhibitor.

\textbf{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 was\emph{trans}
-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 include\emph{trans} -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), \emph{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 \selectlanguage{greek}α-\selectlanguage{english}amidated peptides (Abou-Mohamed et al., 2000;
Ogonowski et al., 1997). The methyl ester prodrug of PBA was
\textasciitilde{}10-fold more effective than PBA in inhibiting the
growth of tumorigenic rat liver epithelial cells (WB-\emph{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 O\textsubscript{2} 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 \textsuperscript{14}C-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.

\textbf{5. What have we learned towards the design of a PAM targeted
therapeutic}

When viewed collectively, patterns emerge from the studies of the PHM
inhibitors and inactivators. Clearly, PHM prefers a free carboxylate
conjugated to a hydrophobic moiety positioned as close as possible to
the penultimate amino acid for the glycine-extended peptide substrates.
Incorporation of a sulfur-containing group that can coordinate with one
of the PHM-bound copper atoms would likely increase binding affinity.
Other possibilities to increase binding affinity in future PHM
inhibitors/inactivators would be to link mimosine (or a mimosine analog)
to a compound that binds at the peptide site to create a bifunctional
inhibitor. The identification of a putative mimosine-binding site
(\textbf{Fig.3C} ) in conjunction with the published PHM structures (W.
A. Francisco, Blackburn, \& Klinman, 2003; Prigge et al., 2004) and the
wealth of structure-activity data for PHM substrates and inhibitors
provide an excellent starting point for \emph{in silico} modeling of
high affinity PHM inhibitors or inactivators. Another possible
bifunctional inhibitor could include a copper-chelator linked to a
compound that binds at the peptide site in PHM. Inhibitors/inactivators
with high affinity unique to PAM could possess a PHM binder linked to
the PAL-specific pyruvate-extended amino acids. Without a structure for
bifunctional PAM, a series of compounds with different length spacers
between the PHM inhibitor and the PAL inhibitor would be required to
define the compound with the highest affinity for PAM. The appropriate
incorporation of mimosine into a PAM inhibitor could yield a
tight-binding trifunctional inhibitor with a group that binds into the
mimosine site of PHM, the peptide site of PHM, and the substrate site of
PAL.

The development of a high affinity PHM (or PAM) inhibitor will encounter
significant hurdles that could hinder clinical use. One concern is
delivery. PHM is found within the lumen of the secretory pathway (Kumar,
Mains, et al., 2016), a challenging site for drug delivery. As discussed
above, the clinical use of a PHM inhibitor is likely dependent upon a
molecular zip code for the secretory system or the surface of specific
cell types. Perhaps an engineered version of the Shiga toxin might
enable the delivery of a high affinity PHM/PAM inhibitor to the
secretory system (Luginbuehl, Meier, Kovar, \& Rohrer, 2018). Another
concern is the diversity of the amidated products produced \emph{in
vivo} by PHM. The inhibition of PHM would produce unselective blocking
of the biosynthesis of many amidated peptides and lipids. One solution
to this concern is not a PHM inhibitor, but the development of inhibitor
that binds selectively and with high affinity to one glycine-extended
substrate, which inhibits the amidation of only one PHM substrate
(Weiss, McIntyre, McLaughlin, \& Merkler, 2006).

The inactivators require at least one \emph{trans} -olefinic bond
positioned \selectlanguage{greek}β- \selectlanguage{english}to a carboxylate for the most efficient inactivation.
Inactivators with the highest affinity have the inactivating moiety
attached to the C-terminus of a peptide or a hydrophobic group like a
phenyl group. The hydrophobic group must be appropriately spaced away
from the inactivating species for the highest affinity, exactly as was
observed for the glycine-extended substrates and the PHM inhibitors.
Again, the available structure-activity data and PHM structures provide
an excellent backdrop for the \emph{in silico} design of high affinity
inactivators. The decoration of such high affinity inactivators with the
appropriate imaging reagent could yield a PHM-specific imaging reagent,
demonstrating that the molecular targeting was in fact successful.
Another application would be decoration with biotin to enable the
profiling of PHM similar to the activity-based profiling strategies
developed by Cravatt et al. (Cravatt, Wright, \& Kozarich, 2008).
However, additional research is required to unravel uncertainties in the
PHM inactivation chemistry to fully exploit the development of a
PHM-specific imaging reagent.

\textbf{6. The bigger picture -- PAM from green algae to human}

PAM and other granule membrane proteins are deposited on the cell
surface during the process of exocytosis (\textbf{Fig.4A} ). Immature
granules contain PAM and soluble cargo proteins. During the maturation
process, cargo proteins are further modified; the granules move from the
Golgi region towards the cell surface and acquire the cytosolic proteins
needed to make them responsive to the right stimuli. Any PAM protein in
the granule membrane appears on the cell surface as the soluble cargo
proteins are released into extracellular space (\textbf{Fig.4A} ,
Exocytosis). Under normal conditions, PAM is rapidly removed from the
plasma membrane (\textbf{Fig.4A} , Endocytosis) and returned to the
secretory granules via a complex series of steps through the endocytic
pathway. Clathrin coated vesicles mediate the removal of PAM from the
cell surface and detailed studies of PAM trafficking in corticotrope
tumor cells have tracked the internalized protein as it moves from early
endosomes into late endosomes (B\selectlanguage{ngerman}äck et al., 2017; Rajagopal, Stone,
Francone, Mains, \& Eipper, 2009). Whether the PAM protein that exits
late endosomes is degraded in lysosomes or is recycled to immature
granules (\textbf{Fig.4A} ) is determined by O-linked sugars attached to
the linker region between PHM and PAL, by a conserved pH-sensitive
region that follows PHMcc and by the phosphorylation status of the PAM
cytosolic domain (Rajagopal et al., 2009; Rao, Zavala, Deb Roy, Mains,
\& Eipper, 2019; K. Vishwanatha, Bäck, Mains, \& Eipper, 2014; K. S.
Vishwanatha, Bäck, Lam, Mains, \& Eipper, 2016). Targeting its O-linked
sugars or its pH-sensitive region could provide a means of preventing
the return of PAM to the secretory pathway.

Compounds that stimulate the secretion of peptide hormones and
neurotransmitters stored in granules increase the amount of PAM protein
delivered to the plasma membrane (\textbf{Fig.4A} , lightning bolt).
Tumor cells often secrete autocrine growth factors, many of which are
amidated peptides (Jimenez et al., 2001). The appearance of PAM on the
surface of these cells provides an opportunity for cell-impermeant PHM
inhibitors or PAM-targeted tags that can be tracked \emph{in vivo} to
bind to its ectodomain and enter the endocytic pathway along with PAM.
Internalization of a PHM inhibitor would be expected to reduce the
synthesis of bioactive amidated growth factors while ectodomain
antibodies or active-site targeted tags might facilitate tumor cell
localization (\textbf{Fig.4A} ). An inactive prodrug might be expected
to bind to PAM while it is exposed on the cell surface, with drug
activation designed to occur in the lower pH environments encountered
during endocytic trafficking. As discussed above, with an understanding
of the structure of bifunctional PAM, the design of dual function drugs
(targeting PHM and PAL) might provide a tool capable of preventing the
endocytic trafficking needed to return PAM to the secretory pathway.
Outside of its active sites, the sequences of insect and human PHM may
be divergent enough to design species specific inhibitors useable as
insecticides.

Phylogenetic studies have suggested several different situations in
which controlling PAM activity or localization could be beneficial.
While the active sites of \emph{C. reinhardtii} PAM (CrPAM) include each
of the residues identified as essential in vertebrate PHM and PAL, this
unicellular green alga does not store peptides in secretory granules
(\textbf{Fig.4B} ). CrPAM is localized to the membranes of the Golgi
complex, as observed in vertebrates, but the presence of two prominent
motile cilia made it clear that CrPAM is also localized to the ciliary
membrane (\textbf{Fig.4B} ) (Kumar, Blaby-Haas, et al., 2016). Unlike
the enzymes used by \emph{C. reinhardtii} to respond to the need for
various nutrients, neither PAM activity nor PAM protein is secreted
through the classical secretory pathway (Kumar et al., 2017; Luxmi et
al., 2019). In vertebrate systems, endoproteolytic cleavages that
separate the PHM and PAL domains from the transmembrane domain result in
their secretion under both basal and stimulated conditions (Kumar,
Mains, et al., 2016). During vegetative growth, \emph{C. reinhardtii}
rely on bioactive ectosomes released from their cilia (\textbf{Fig.4B} ,
Inset) to destroy the mother cell wall (Wood, Huang, Diener, \&
Rosenbaum, 2013). Ciliary ectosomes also play a key role during sexual
reproduction in \emph{C. reinhardtii} (M. Cao et al., 2015). Active
CrPAM is present in mating ectosomes, along with its amidated products,
one of which acts as a chemomodulator (Luxmi et al., 2019)
(\textbf{Fig.4B} ). PAM was subsequently identified in the ciliary
membranes of vertebrate motile and sensory cilia (Kumar et al., 2017).
The formation of ciliary ectosomes (\textbf{Fig.4B} ) is topologically
similar to the formation of intraluminal vesicles in the endocytic
pathway (\textbf{Fig.4C} ). Fusion of a multivesicular body containing
ILVs results in their release as exosomes (\textbf{Fig.4C} ). PAM has
been identified in intraluminal vesicles (Bäck et al., 2017) and in
exosomes isolated from human saliva and urine (Gonzalez-Begne et al.,
2009; Principe et al., 2013; Z. Wang, Hill, Luther, Hachey, \& Schey,
2012). Much remains to be learned about the role played by
ectosomes/exosomes, but the presence of PAM in these structures offers a
means of isolating and characterizing that subset.

When PAM expression was reduced in \emph{C. reinhardtii} , the cells
were unable to form cilia (\textbf{Fig.4D} ) (Kumar et al., 2017).
Instead of their normal pair of motile 12 \selectlanguage{greek}µ\selectlanguage{english}m long cilia (\textbf{Fig.4B}
), PAM knockdown cells had ciliary stubs containing microtubule
fragments. The ciliary axoneme did not form and the appearance of the
transition zone was altered. In humans, fully 5\% of the genome is
thought to be involved in the formation and function of cilia and
ciliopathies are associated with many congenital diseases including
obesity, blindness and kidney disease (Engle, Bansal, Antonellis, \&
Berbari, 2021; Leroy et al., 2021; Nager et al., 2017). The effects of
reducing PAM levels on ciliogenesis are species specific, but occur in
planaria, zebrafish and mouse (\textbf{Fig.4D} ). The hypothalamic
neurons that produce proopiomelanocortin must have cilia and the
melanocortin 4 receptor must be localized to those cilia in order to
regulate appetite normally (Y. Wang et al., 2021). Much remains to be
learned about the sensory and signaling roles of cilia and tools that
allow the manipulation of ciliary PAM would be of great utility in these
studies.

It is now known that the PAM protein has several non-catalytic functions
(\textbf{Fig.4E} ). Regulated intramembrane proteolysis of PAM releases
a soluble fragment of its cytosolic domain (sfCD). Nuclear accumulation
of sfCD depends on its phosphorylation status and is thought to
contribute to the ability of PAM to alter gene expression in a
tissue-specific manner (Rajagopal et al., 2009). In atrial myocytes,
which express PAM at levels higher than those observed in other cells,
the PAM protein (whether active or inactive) plays an essential role in
the formation of atrial granules, which store natriuretic peptides
(B\selectlanguage{ngerman}äck, Luxmi, Powers, Mains, \& Eipper, 2020). Instead of its catalytic
activity, the ability of PAM to function as a re-usable luminal cargo
receptor for proatrial natriuretic peptides early in the secretory
pathway is thought to be essential to its ability to support
granulogenesis.

\textbf{Acknowledgments}

We thank the many lab members and colleagues over several decades whose
insights and~hard work contributed to these studies. Supported by: NIH
R01-DK032948 and R01-DK032949 (BAE, REM); NIH R01-GM051293 (BAE, Stephen
M. King); The Daniel Schwartzberg Fund (BAE, REM); NIH R21-GM140390 and
R15-GM073659 (DJM); Eppley Foundation for Research (DJM); Milheim
Foundation for Cancer Research (DJM); Unigene Laboratories, Inc.
(DJM).\emph{Figure Legends}

\textbf{Figure 1. Peptide processing basics, the amidation reaction and
amidation enzymes.} \textbf{A.} The preprotein peptide precursor is
processed by endoproteases (prohormone convertases such as PCSK1, PCSK2,
PCSK3) and an exoprotease (such as CPE or CPD) to present to PAM the
immediate precursor (peptide-Gly), which yields the final amidated
peptide product. \textbf{B.} The two-step PAM reaction. Step 1 is
performed by the first enzyme, PHM, and Step 2 is performed by PAL. The
two copper residues bound to PHM are reduced by ascorbate, peptide and
dioxygen bind to the reduced enzyme, a proton is abstracted and the
peptide is hydroxylated by PHM. The N-C bond is then cleaved by PAL,
liberating the \selectlanguage{greek}α-\selectlanguage{english}amidated peptide plus glyoxalate. \textbf{C.} Proteins
used for enzymology and structural determination. In mammals, two major
forms of PAM are the bifunctional membrane enzymes PAM1 and PAM2, each
with a transmembrane domain (TMD) and short cytoplasmic domain (CD). The
proteins used thus far for structural analyses are the recombinant
catalytic cores, PHMcc and PALcc. The reactions have been studied with
purified natural proteins from various sources, recombinant pure rat
PHMcc and PALcc, and with two pure recombinant bifunctional proteins,
rat PHM-PAL820s and Type A a-AE. References given in the text. Residues
(using NP\_037132.2): PHMcc (42-356), PALcc (498-820, lacking
N-glycosylation {[}S767A{]}), PHM-PAL820s (42-820 lacking Exon 16
{[}residues 393-498{]}, lacking N-glycosylation {[}S767A{]}), Type A PAM
(27-820 lacking Exon 16).

\textbf{Figure 2. The Reactions Catalyzed by PHM and PAL.} Bifunctional
PAM is compromised of the two separate catalytic units, PHM and PAL
(Reaction A). Steps 4 and 5 in reaction A represent a collection of
steps in the PHM mechanism (Cowley et al., 2016; Prigge et al., 2000; Wu
et al., 2019). Reactions B and C catalyzed by PHM, any involvement of
PAL in reactions B and C have not been specifically addressed.
The\emph{S} -dealkylation reaction, shown in reaction B, is consistent
with the finding of glyoxylate as a minor product during the
sulfoxidation reaction. A sulfoxide/glyoxylate ratio of 8 was reported,
but no mercaptan was found (A.G. Katopodis \& S.W. May, 1990). The
formation of glyoxylate from the imino-oxy acetate (reaction D, bottom
reaction) is PAL-independent.

\textbf{Figure 3. Ligands that Bind to PHM and PAL.}
\textbf{A.}Structures for the inhibitors and activators discussed in
this review.\textbf{B.} \emph{In silico} model for the interaction of
PHMcc with\emph{N} -hydrocinnamoyl-L-Phe-L-homocysteine,
IC\textsubscript{50} = 10 nM (compound \#22 from (Erion et al., 1994)).
\textbf{C.} \emph{In silico} model for the interaction of PHMcc and
mimosine. \textbf{D.}\emph{In silico} model for the interaction better
PHMcc and mimosine or ascorbate. \textbf{E.} \emph{In silico} model of
the interaction of PHMcc with N-\selectlanguage{greek}α-\selectlanguage{english}acetyl-3,5-diodotyrosylglycine and
ascorbate. Our model is based on the published model of the PHMcc
complexed with the same dipeptide (Prigge et al., 2000). The copper
atoms are shown in brown (B-E) and the PHMcc backbone is either gray (B)
or light green (C-E). For Panels C-E, the amino acid ligands for the
copper atoms are in blue and binding site amino acid side chains are in
dark green. The colors for the ligands are as follows: \emph{N}
-hydrocinnamoyl-L-Phe-L-Phe is purple (B), mimosine is purple (C and D),
ascorbate is brownish-yellow (D and E), and \emph{N}
-\selectlanguage{greek}α-\selectlanguage{english}acetyl-3,5-diiodotyrosylglycine is gray (E). The \emph{in silico}
models were generated using AutoDock Vina.\emph{N}
-Hydrocinnamoyl-L-Phe-L-homocysteine was docked using the flexible side
chain method, with the ligand covalently attached and then modeled as a
flexible residue (Bianco, Forli, Goodsell, \& Olson, 2016).

\textbf{Figure 4. A.} PAM trafficking in neuroendocrine cells. As newly
synthesized PAM and soluble cargo proteins exit the \emph{trans} -Golgi,
they accumulate in immature secretory granules. Granule maturation
involves vesicular trafficking and acquisition of the cytosolic proteins
needed to respond to secretagogues. Upon fusion of the secretory granule
membrane with the plasma membrane, soluble content proteins are released
and membrane PAM appears on the cell surface. Clathrin-mediated
endocytosis means that less than 5\% of the PAM protein in a cell
typically resides on the plasma membrane. PAM retrieved from the cell
surface can be degraded or returned to the secretory pathway for
re-use.\textbf{B.} In \emph{C. reinhardtii} , PAM is localized to the
Golgi Complex, small vesicular structures and the ciliary membrane. The
ciliary budding process that generates ectosomes is illustrated, with
the localization of CrPAM and one of its amidated products
(Cre03.g20450) illustrated (Luxmi et al., 2018; Luxmi et al.,
2019).\textbf{C.} The movement of PAM through late endosomes and into
the intraluminal vesicles (ILVs) that form in multivesicular bodies
(MVBs) was determined using ectodomain antibodies (B\selectlanguage{ngerman}äck et al., 2017;
Rajagopal, Mains, \& Eipper, 2012). Upon fusion with the plasma
membrane, PAM-containing exosomes are released. \textbf{D.} The
species-specific roles of PAM in ciliogenesis are summarized.\textbf{E.}
The non-catalytic effects of PAM are summarized. The ability of PAM to
alter gene expression is thought to require the generation of sfCD
through \selectlanguage{greek}γ-\selectlanguage{english}secretase-catalyzed regulated intramembrane proteolysis (RIP)
(Rajagopal, Stone, Mains, \& Eipper, 2010). Neither the ability of PAM
to support the formation of atrial granules or its ability to interact
with actin require its catalytic activity.\emph{}

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