Biomedical Engineering Reference
In-Depth Information Active-Site Architecture and Substrate Specificity
PfA-M17 possesses a severely restricted specificity for N-terminally exposed
Leu; Ala and Phe substrates are cleaved poorly, and substrates containing an
Arg, Val, Gly, Ile, Glu, or Asp in the P1 position are not cleaved at all. 31
Despite a preference for P1 leucine residues, the PfA-M17 has a more restricted
specificity than other characterized LAP enzymes. The structural reason for
this is immediately apparent from the architecture of the active site 42 . The PfA-
M17 active site cleft measures 1.8 nm by 1.8 nm. The S1 pocket is very narrow
and lined at its entrance by substantially hydrophobic residues including
(Met 392 , Met 396 , Phe 398 , Thr 486 , Gly 489 , Leu 492 , and Phe 583 ). There are no
residues capable of forming hydrogen bonds with a polar P1 residue in a
The S1 0 pocket of PfA-M17 does not form a classic pocket shape. It has an
open geometry and orientates the P1 0 residue into the open interior of the active
site cavity of the biological hexamer. On one side of the pocket, charged and
polar residues (Asn 457 , Arg 463 , Ile 547 , Ser 548,550 , Lys 552 , and Ser 554 ) could
contribute hydrogen-bonding partners to a variety of amino acids or indeed
other chemical moieties selected for desirable qualities such as pharmaco-
kinetically favorable, enhanced anities and/or mimetic groups. Also, the fact
that the P1 0 is orientated away from the active site means that future inhibitors/
lead compounds could be made chemically larger than dipeptide analogs. The
limiting factor of size would be entry into the active site cavity.
7.3 Structure-Activity-Relationship Data and
Drug Design
Although the M1 and M17 protease superfamilies are large and divergent, the
two proteases utilise a common catalytic mechanism by the coordination of one
or two cations in the active site to activate water for nucleophilic attack of a
peptide or protein substrate. To exploit this common core mechanism, several
classic inhibitor scaffolds have been developed to target the neutral amino-
peptidase family including, most prominently, phosphinic acids, 45,46 hydr-
oxamic acids, 47 and the bestatin family. 22,28,29,46 The many inhibitors of
aminopeptidases have been extensively reviewed recently. 48
7.3.1 Current Inhibitors of the Neutral Aminopeptidases
Two inhibitors of both PfA-M1 and PfA-M17 that have been structurally well
characterized are bestatin and Compound 4 (Co4; phosphinic dipeptide
analog). 30,42,46 In vitro, both bestatin and Co4 are far superior inhibitors of
PfA-M17 than PfA-M1. Analysis of the X-ray crystal structures of both
enzymes bound to these two inhibitors reveals that they each make far more
extensive interactions with the PfA-M17 active site than with PfA-M1. Both
inhibitors coordinate an active site Zn 21 atom (equivalent to the site 1 metal in
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