view release on metacpan or  search on metacpan

t/data/macroman/11042188.macroman  view on Meta::CPAN

   significant loss in potency, again emphasizing the contribution to
   binding by appropriate positioning of the large benzene ring of the
   side chain of Phe^30. In contrast, replacement of Val^26 by Ala did not
   diminish inhibitory potency. Rather, it appeared to improve the binding
   interaction at pH 3.1 marginally (peptide 24, Table [141]IV).
   Replacement of the -CH[3] side chain of Ala with the -CH[2]-COOH side
   chain of an Asp at position 26 diminished the inhibitory potency at pH
   4.7 by about 70-fold (compare peptides 25 and 24, Table [142]IV),
   commensurate with the introduction of a hydrophilic side chain into a
   hydrophobic environment. However, theK [i] value measured at pH 3.1 for
   peptide 25 was tighter than that derived at pH 4.7 (Table [143]IV). Of
   all the inhibitors listed in Tables [144]I, [145]II, and [146]IV, this
   was the only occasion when such an effect was observed and most likely
   is a reflection of the Asp side chain in its protonated and therefore
   uncharged form being less unfavorable in its contact with the
   hydrophobic environment offered by the enzyme.

   The double mutant peptide carrying the V26D/F30K substitutions was
   completely ineffective as an inhibitor (peptide 26, Table [147]IV),
   indicating that introduction of two hydrophilic, charged residues was
   highly unfavorable since there are no H-bond partners available in the
   enzyme to compensate for desolvation of the two side chain functions.
   However, amphipathic helices are often stabilized by electrostatic
   interactions between residues at positions i andi + 4 ([148]23) and,
   indeed exactly such a salt bridge is present between the Lys^24 and
   Asp^28 residues on the hydrophilic face of the IA[3] helix when
   complexed with its target proteinase (Fig. [149]1). However, the
   attempt to encourage Asp^26 and Lys^30 to interact with one another to
   form an additional salt bridge in the V26D/F30K double mutant peptide
   was clearly not tolerated on the hydrophobic face of the amphipathic
   helix in the active site cleft of the enzyme. A further mutant was also
   constructed in which the sequence of
   ∼Ser^27-Asp^28-Ala^29-Phe^30-Lys^31-Lys^32∼ was shuffled to
   ∼Lys^27-Ala^28-Asp^29-Lys^30-Phe^31-Ser^32∼ in the protein form of the
   IA3 inhibitor (Mix in Table [150]I). In this arrangement, Val^26 was
   retained but the salt bridge between Lys^24 and Asp^28 on the
   hydrophilic face of the helix was disrupted and the crucial Phe^30
   residue was replaced by lysine, as in peptide 23. The resultant mutant
   protein (Mix, in Table [151]I) was purified to homogeneity from E.
   coliand found to have a K [i] value at pH 4.7 comparable to that
   observed for the single F30K mutant peptide (peptide 23, Table[152]IV).
   This might be interpreted to indicate that the salt bridge interaction
   between Lys^24 and Asp^28 on the hydrophilic surface of the IA[3] helix
   is, not unexpectedly, weak.

 ¤5¤ Central Residues in the Inhibitor Helix ¤5¤

   The ÒcenterpieceÓ of the inhibitory 2Ð34 residues of IA[3] is the
   ∼Lys^18-Leu^19-X-X-Asp^22∼ sequence. In the three crystal structures of
   the K24M and K31M/K32M mutant proteins and peptide 1 forms of IA3
   complexed with proteinase A, the ε-NH[2] group of Lys^18 of the
   inhibitor hydrogen bonds to one of the carboxyl oxygens of Asp^32 of
   the enzyme (Fig. [153]3). This is one of the two catalytic Asp residues
   that operate the catalytic mechanism of all aspartic proteinases
   ([154]24). The ε-NH[2] group of Lys^18 also hydrogen bonds to one of
   the carboxyl oxygens of the side chain of Asp^22 in the IA[3]
   inhibitory sequence (Fig. [155]3). The other oxygen of the side chain
   COOH of Asp^22 hydrogen bonds to the phenolic OH group of Tyr^75 in the
   enzyme, a residue that is totally conserved in all eukaryotic aspartic
   proteinases and which is positioned almost at the tip of the β-hairpin
   loop or flap that overlays the active site cleft in these enzymes. A
   network of interactions thus cross-links these charged residues of
   IA[3]with the catalytically essential and structurally conserved
   residues of the target enzyme (Fig. [156]3). When the charged side
   chain of Asp^22 in the full-length protein form of IA[3] was replaced
   with the hydrophobic but otherwise almost isosteric side chain of a
   leucine residue, the purified D22L mutant protein had a slightly
   reduced potency both at pH 3.1 and 4.7 (Table [157]I) but nevertheless
   was still an effective inhibitor.
   [158]Figure 3
   View larger version:
     * [159]In this window
     * [160]In a new window

     * [161]Download as PowerPoint Slide

   Figure 3

   Stereo representation of the interactions made by the
   ∼Lys^18-Leu^19-X-X-Asp^22∼ centerpiece residues of IA[3] in the
   vicinity of the active site of proteinase A. Proteinase A residues are
   ingreen with the catalytic water molecule depicted as ablue sphere. The
   side chains of Lys^18, Leu^19, and Asp^22 plus the relevant segment of
   the IA[3] helix backbone are in brown. Hydrogen bonding distances are
   shown.

   However, when Lys^18 was changed to Met in concert with the D22L
   mutation, the resultant inhibitor (K18M/D22L, Table [162]I) was an
   extremely potent inhibitor. For the first time in all of these studies,
   it was not possible to derive an accurateK [i] value at pH 3.1 because
   the protein-protein interaction was so tight. This may be a reflection
   of the increased propensity of Met and Leu residues to be accommodated
   within a helical conformation by comparison with their wild-type Lys
   and Asp counterparts. Alternatively, this may be a further indication
   of the importance of hydrophobic contributions to binding strength.
   Modeling studies suggest that the side chain of a methionine at
   position 18 in the IA[3] sequence is surrounded by the hydrophobic side
   chains of Ile^30, Tyr^75, Thr^111, Phe^112, Phe^117, and Ile^120 of
   proteinase A, as well as the newly introduced side chain of Leu^22 in
   the K18M/D22L double mutant inhibitor. The side chain of Leu^22 can
   make potential, favorable interactions with C-β of Ser^35 and the side
   chains of Ile^73 and Tyr^75 from the flap of proteinase A, as well as
   with the Met^18 and Val^25 residues of the IA[3] inhibitory sequence.

   The importance of hydrophobic interactions was further corroborated
   when the Leu^19 residue within the ∼Lys^18-Leu^19-X-X-Asp^22∼
   centerpiece was changed to Ala in the peptide form of IA[3]. The
   resultant peptide (peptide 27, Table [163]IV) was no longer an
   inhibitor at pH 3.1 and an apparent inhibition constant of 700 nm was
   estimated at pH 4.7. This loss in potency (compare peptides 27 and 9,
   Table [164]IV) was the largest observed for any single amino acid
   replacement. However, when peptide 27 was incubated with proteinase A
   for 16 h at pH 4.7 and 37 ¡C at a molar ratio of 40:1, it was cleaved
   as a substrate (as monitored by reverse phase fast protein liquid
   chromatography, not shown). Peptide 27 is thus a good alternative
   substrate at pH 4.7 and was giving only an apparent inhibition of the
   activity of proteinase A toward the chromogenic substrate used in the
   inhibition assays. The deletion of the terminal isopropyl moiety of the
   leucine side chain thus converted a highly potent polypeptide inhibitor
   into a regular substrate of proteinase A by decreasing affinity to the
   target enzyme and possibly by reducing internal helix stability.