The selectivity of action of the aspartic-proteinase inhibitor IA3 from yeast (<i>Saccharomyces cerevisiae</i>)

Dreyer, Thomas; Valler, Martin J.; Kay, John; Charlton, Peter; Dunn, Ben M.

doi:10.1042/bj2310777

Cited by 30 publications

(27 citation statements)

References 20 publications

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“…The K i value determined at pH 3.1 for the inhibition of yeast proteinase A by this C-terminal tagged, wild-type recombinant protein (wild-type in Table I) was comparable to that reported previously at the same pH for the naturally occurring protein purified from S. cerevisiae (14). It is readily apparent then that the introduction of the extra ϳLeu-Glu-His 6 residues at the C terminus of the recombinant inhibitor did not have any significant detrimental effect on inhibitory potency.…”

Section: Interaction Of Protein/peptide Forms Of Ia 3 With Target Andsupporting

confidence: 61%

“…Kinetic Measurements and Peptide Cleavage-Inhibition assays were conducted at pH 3.1 and 4.7 as described previously (12,14) using Lys-Pro-Ile-Glu-Phe*NitroPhe-Arg-Leu (where the asterisk indicates the scissile peptide bond) as substrate for all enzymes except yapsin 1 (15). For this enzyme, the substrate used was ACTH (residues 1-39).…”

Section: Methodsmentioning

confidence: 99%

“…A synthetic peptide which spanned residues 2-34 of the IA 3 sequence (Peptide 1 in Table II) was found to have inhibitory potency against yeast proteinase A at pH 3.1 and 4.7 compa-rable to those of the naturally occurring and wild-type recombinant protein forms of IA 3 , described previously (12,14). In contrast, peptide 1, at a concentration of 2 M, had no significant ability to inhibit any one of a number of other aspartic proteinases from a wide range of other species (Table III).…”

Section: Interaction Of Protein/peptide Forms Of Ia 3 With Target Andmentioning

confidence: 99%

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The Potency and Specificity of the Interaction between the IA3 Inhibitor and Its Target Aspartic Proteinase fromSaccharomyces cerevisiae

Phylip¹,

Lees²,

Brownsey³

et al. 2001

Journal of Biological Chemistry

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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 ␣-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 typecast 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, ␤-site Alzheimer's precursor protein cleavage enzyme and ␤-site Alzheimer's precursor protein cleavage enzyme 2 (1, 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 (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 (4); a 17-kDa inhibitor of pepsin and cathepsin E from the parasite, Ascaris lumbricoides (5); proteins from plants such as potato, tomato, and squash (6, 7), and a pluripotent inhibitor from sea anemone of cysteine proteinases as well as cathepsin D (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 (9). The complete sequence of this 68-residue inhibitor has been elucidated (10, 11) and the inhibitory activity of IA 3 has been shown to reside within the N-terminal half of the molecule (10, 12). ...

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Section: Interaction Of Protein/peptide Forms Of Ia 3 With Target Andsupporting

confidence: 61%

Section: Methodsmentioning

confidence: 99%

Section: Interaction Of Protein/peptide Forms Of Ia 3 With Target Andmentioning

confidence: 99%

See 1 more Smart Citation

The Potency and Specificity of the Interaction between the IA3 Inhibitor and Its Target Aspartic Proteinase fromSaccharomyces cerevisiae

Phylip¹,

Lees²,

Brownsey³

et al. 2001

Journal of Biological Chemistry

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“…Similarly, the naturally occurring protein inhibitors of pepsin/ cathepsin E and yeast proteinase A from the parasitic worm A. lumbricoides (33) and S. cerevisiae (34), respectively, did not inhibit the recombinant cyprosin to any significant extent (IC 50 Ͼ Ͼ 2,000 nM).…”

Section: Fig 4 Schematic Representation Of Processing Events In Pmentioning

confidence: 99%

Processing, Activity, and Inhibition of Recombinant Cyprosin, an Aspartic Proteinase from Cardoon (Cynara cardunculus)

White

Cordeiro²,

Arnold

et al. 1999

Journal of Biological Chemistry

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The cDNA encoding the precursor of an aspartic proteinase from the flowers of the cardoon, Cynara cardunculus, was expressed in Pichia pastoris, and the recombinant, mature cyprosin that accumulated in the culture medium was purified and characterized. The resultant mixture of microheterogeneous forms was shown to consist of glycosylated heavy chains (34 or 32 kDa) plus associated light chains with molecular weights in the region of 14,000 -18,000, resulting from excision of most, but not all, of the 104 residues contributed by the unique region known as the plant specific insert. SDS-polyacrylamide gel electrophoresis under non-reducing conditions indicated that disulfide bonding held the heavy and light chains together in the heterodimeric enzyme forms. In contrast, when a construct was expressed in which the nucleotides encoding the 104 residues of the plant specific insert were deleted, the inactive, unprocessed precursor form (procyprosin) accumulated, indicating that the plant-specific insert has a role in ensuring that the nascent polypeptide is folded properly and rendered capable of being activated to generate mature, active proteinase. Kinetic parameters were derived for the hydrolysis of a synthetic peptide substrate by wildtype, recombinant cyprosin at a variety of pH and temperature values and the subsite requirements of the enzyme were mapped using a systematic series of synthetic inhibitors. The significance is discussed of the susceptibility of cyprosin to inhibitors of human immunodeficiency virus proteinase and particularly of renin, some of which were found to have subnanomolar potencies against the plant enzyme.

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“…Moreover, they are involved in the replication and survival of many pathways of microorganisms [15 -17]. Aspartic proteinases are found in vertebrates, fungi, plants and retroviruses [15,16,18,19]. Some important examples of aspartic proteinases are pepsin [3 -6], the secreted aspartic proteinases (SAPs) from Candida albicans [20], the HIV proteinase [27] and the yeast proteinase A [10].…”

Section: Introductionmentioning

confidence: 99%

Kinetic analysis of a general model of activation of aspartic proteinase zymogens involving a reversible inhibitor. I. Kinetic analysis

Muñoz-López

Sotos-Lomas

Garde

et al. 2007

Journal of Enzyme Inhibition and Medicinal Chemistry

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The selectivity of action of the aspartic-proteinase inhibitor IA3 from yeast (Saccharomyces cerevisiae)

Cited by 30 publications

References 20 publications

The Potency and Specificity of the Interaction between the IA3 Inhibitor and Its Target Aspartic Proteinase fromSaccharomyces cerevisiae

The Potency and Specificity of the Interaction between the IA3 Inhibitor and Its Target Aspartic Proteinase fromSaccharomyces cerevisiae

Processing, Activity, and Inhibition of Recombinant Cyprosin, an Aspartic Proteinase from Cardoon (Cynara cardunculus)

Kinetic analysis of a general model of activation of aspartic proteinase zymogens involving a reversible inhibitor. I. Kinetic analysis

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