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   [1]The Journal of Biological Chemistry

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 ¤1¤ The Potency and Specificity of the Interaction between the IA[3]
Inhibitor and Its Target Aspartic Proteinase fromSaccharomyces
cerevisiae[15]* ¤1¤

    1. [16]Lowri H. Phylip[17]tOa,
    2. [18]Wendy E. Lees[19]tOa[20]FNb,
    3. [21]Brian G. Brownsey[22]tOc,
    4. [23]Daniel Bur[24]tOd,
    5. [25]Ben M. Dunn[26]tOe,
    6. [27]Jakob R. Winther[28]tOf,
    7. [29]Alla Gustchina[30]tOg,
    8. [31]Mi Li[32]tOg[33]tOh,
    9. [34]Terry Copeland[35]i,
   10. [36]Alexander Wlodawer[37]tOg and
   11. [38]John Kay[39]tOa[40]FNj

    1.


    From the ^tOaSchool of Biosciences, Cardiff University, P. O. Box
    911, Cardiff CF10 3US, Wales, United Kingdom, the^tOcDepartment of
    Medicine, University of Wales College of Medicine, Cardiff CF14 4XN,
    Wales, United Kingdom, ^tOdF. Hoffmann La Roche AG, CH-4070 Basel,
    Switzerland, the ^tOeDepartment of Biochemistry & Molecular Biology,
    University of Florida College of Medicine, Gainesville, Florida
    32610, the ^tOfDepartment of Yeast Genetics, Carlsberg Laboratory,
    DK-2500, Copenhagen Valby, Denmark, the^tOgProtein Structure Section,
    Macromolecular Crystallography Laboratory, National Cancer Institute,
    Frederick, Maryland 21702, the^tOhIntramural Research Support
    Program, SAIC Frederick, National Cancer Institute, Frederick,
    Maryland 21702, and the ^iProgram Core, DBS, National Cancer
    Institute, Frederick, Maryland 21702


   [41]Next Section

 ¤2¤ Abstract ¤2¤

   The yeast IA[3] polypeptide consists of only 68 residues, and the free
   inhibitor has little intrinsic secondary structure. IA[3] showed
   subnanomolar potency toward its target, proteinase A from Saccharomyces
   cerevisiae, and did not inhibit any of a large number of aspartic
   proteinases with similar sequences/structures from a wide variety of
   other species. Systematic truncation and mutagenesis of the IA[3]
   polypeptide revealed that the inhibitory activity is located in the
   N-terminal half of the sequence. Crystal structures of different forms
   of IA[3] complexed with proteinase A showed that residues in the
   N-terminal half of the IA[3] sequence became ordered and formed an
   almost perfect α-helix in the active site of the enzyme. This potent,
   specific interaction was directed primarily by hydrophobic interactions
   made by three key features in the inhibitory sequence. Whereas IA[3]
   was cut as a substrate by the nontarget aspartic proteinases, it was
   not cleaved by proteinase A. The random coil IA[3] polypeptide escapes
   cleavage by being stabilized in a helical conformation upon interaction
   with the active site of proteinase A. This results, paradoxically, in
   potent selective inhibition of the target enzyme.

   Aspartic proteinases participate in a variety of physiological
   processes, and the onset of pathological conditions such as
   hypertension, gastric ulcers, and neoplastic diseases may be related to
   changes in the levels of their activity. Members of this proteinase
   family, e.g. renin, pepsin, cathepsin D, and human immunodeficiency
   virus-proteinase are generally type-cast on the basis of their
   susceptibility to inhibition by naturally occurring, small molecule
   inhibitors such as the acylated pentapeptides, isovaleryl- and
   acetyl-pepstatin. However, the two most recently identified human
   aspartic proteinases, β-site Alzheimer's precursor protein cleavage
   enzyme and β-site Alzheimer's precursor protein cleavage enzyme 2
   ([42]1, [43]2), are not inhibited by this classical type of inhibitor
   of this family of enzymes. Pepstatins are metabolic products produced
   by various species of actinomycetes and, as such, are not themselves
   gene-encoded. Protein inhibitors of aspartic proteinases are relatively
   uncommon and are found in only a few specialized locations ([44]3).
   Examples include renin-binding protein in mammalian kidneys which
   intriguingly has now itself been identified to be the
   enzyme,N-acetyl-d-glucosamine-2-epimerase ([45]4); a 17-kDa inhibitor
   of pepsin and cathepsin E from the parasite, Ascaris lumbricoides
   ([46]5); proteins from plants such as potato, tomato, and squash
   ([47]6, [48]7), and a pluripotent inhibitor from sea anemone of
   cysteine proteinases as well as cathepsin D ([49]8).

   The IA[3] polypeptide in yeast is an 8-kDa inhibitor of the vacuolar
   aspartic proteinase (proteinase A or saccharopepsin) that was initially
   described by Holzer and co-workers ([50]9). The complete sequence of
   this 68-residue inhibitor has been elucidated ([51]10, [52]11) and the
   inhibitory activity of IA[3] has been shown to reside within the
   N-terminal half of the molecule ([53]10, [54]12). We have recently
   solved the structure of the IA[3]-proteinase A complex ([55]12),