Biomedical Engineering Reference
In-Depth Information
6.3. FUNCTIONAL PROPERTIES
6.3.1. Mechanism of Transport
POT transporters use a proton gradient and the membrane potential as the main driving
forces, as reviewed previously
9
,
17
,
70
,
71
, to mediate the uptake of peptides or peptide-
based drugs. In bacteria, yeast, and plant cells, the proton gradient driving force is
supplied primarily by membrane ATPases,
17
while in mammalian cells it is generally
provided by electroneutral proton-cation exchangers (e.g., Na
+
/H
+
antiporters).
72
,
73
For example, PepT1-mediated intestinal absorption of peptide-based substrates occurs
in an asymmetrical manner, with the proton gradient generally considered to be the
predominant force influencing transport in the upper small intestine.
Further studies have demonstrated that charged substrates present different binding
affinities based on their degree of ionization. Di- and tripeptides, and peptidomimetic
compounds with no net charge at the site of absorption are best transferred across
the cell membranes by PepT-like transporters.
74
-
78
PepT substrates share the same
substrate-binding site regardless of substrate charge.
79
,
80
Proton coupling occurs in
the H
+
-binding site of PepT1, where a H
+
is bound prior to anionic or neutral sub-
strate uptake but is not required for cationic substrates.
71
Irie et al. have developed a
computational model to illustrate the H
+
-coupled substrate transport of neutral and
charged molecules, establishing a PepT1 mechanistic model based on two assump-
tions: (1) H
+
binds not only to the H
+
-binding site but also to the substrate-binding
site, and (2) H
+
at the substrate-binding site inhibits the interaction of neutral and
cationic substrates but is necessary for that of anionic substrates.
81
6.3.2. Molecular Requirements for Substrate Recognition and Transport
Some molecular requirements on the structure of PepT-like transporters have been
recognized as essential for substrate recognition and transport. Site-directed mutage-
nesis analyses of single amino acids located within different transmembrane domains
(TMD2, TMD4, and TMD5) have shown that Y56, Y64, Y167, N171, and S174
residues modify or inactivate PepT-like transport activity and/or substrate binding
completely.
82
-
85
Other studies have shown that mutations on W294 and E595 re-
duced Gly-Sar uptake significantly.
82
Sequence alignments of PepTs have shown the presence of conserved histidyl
residues (H57, H121, and H260). Experimental evidence suggests that the histidyl
residue H57 is involved in H
+
-binding and is essential for peptide transport activity.
86
H121 is involved in substrate recognition, while the role of H260 remains to be
elucidated.
84
In hPepT2, H87 has been shown to be absolutely essential to maintain
transport activity.
87
Doring et al. demonstrated that the N-terminal region of the protein (TMD1
to TMD9) confers all the phenotypic characteristics, while studies utilizing
PepT1/PepT2 chimeras suggest that the first 400 residues (TMD1 to TMD6) contain
the substrate-binding pocket and the region that determines pH dependence.
88
-
90
Sup-
porting these findings, Doring et al. further demonstrated that a section between TMD2
and TMD3 (amino acid residues 60 to 91) plays a significant role in the pH-dependent
Search WWH ::
Custom Search