The long term objective of this project is to understand the individual molecular interactions responsible for tight binding, specificity and efficient catalysis in the Aspartic Proteinase class of enzymes. These proteolytic enzymes are found in a wide diversity of organisms and play important roles in many physiological processes: human enzymes pepsin and gastricsin in digestion; renin in regulation of blood pressure; and cathepsin D in intracellular protein turnover and in proenzyme and prohormone processing. The retroviral aspartic proteinases are indispensable for the virus life cycle as they process precursor polyproteins into individual functional entities. Crystallographic analysis has shown that these enzymes share a common three-dimensional structure with an extended binding cleft capable of interacting with seven to eight amino acid residues and situated at the interface between the N-terminal and C-terminal domains in the eukaryotic enzymes and at the dimer interface in the retroviral systems. This cleft interacts with ligands in a common fashion. A remarkably similar pattern of hydrogen bonding to the backbone of bound peptides has been observed. However, specific interactions between individual amino acid side chains in the various enzyme subsites are different. Each enzyme, therefore, has subtly different specificity based on binding at individual """"""""secondary: subsites. This program presents a combined approach in which synthesis of new oligopeptide substrate or inhibitor sequences will be joined with protein engineering, expression and purification of recombinant enzymes to explore the nature of the multiple interactions occurring in the cleft. Enzyme residues identified from crystal structures as being located in a specific subsite will be altered to test specific hypotheses on binding interactions. The mutant proteins will be thoroughly characterized. The kinetic properties of the enzymes will be studied using a chromogenic assay with a series of peptide substrates with sequence changes in the position of the substrate complementary to the change in the enzyme subsite. By examination of a group of inhibitors with regular changes in sequence at the specific position under examination additional information will be obtained to test specific structure-function hypotheses. The information gained in this study will add to the understanding of enzyme-substrate interactions in general and will contribute to the design of selective, therapeutically useful enzyme inhibitors for the Asparatic Proteinase family.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK018865-16A2
Application #
3226172
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1978-06-01
Project End
1996-12-31
Budget Start
1993-01-01
Budget End
1993-12-31
Support Year
16
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Florida
Department
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Janka, Linda; Clemente, Jose; Vaiana, N et al. (2008) Targeting the plasmepsin 4 orthologs of Plasmodium sp. with ""double drug"" inhibitors. Protein Pept Lett 15:868-73
Moose, Rebecca E; Clemente, Jose C; Jackson, Larry R et al. (2007) Analysis of binding interactions of pepsin inhibitor-3 to mammalian and malarial aspartic proteases. Biochemistry 46:14198-205
Clemente, Jose C; Govindasamy, Lakshmanan; Madabushi, Amrita et al. (2006) Structure of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae bound to an allophenylnorstatine-based inhibitor. Acta Crystallogr D Biol Crystallogr 62:246-52
Goldfarb, Nathan E; Lam, Minh T; Bose, Arjo K et al. (2005) Electrostatic switches that mediate the pH-dependent conformational change of ""short"" recombinant human pseudocathepsin D. Biochemistry 44:15725-33
Beyer, Bret B; Johnson, Jodie V; Chung, Alfred Y et al. (2005) Active-site specificity of digestive aspartic peptidases from the four species of Plasmodium that infect humans using chromogenic combinatorial peptide libraries. Biochemistry 44:1768-79
Madabushi, Amrita; Chakraborty, Sibani; Fisher, S Zoe et al. (2005) Crystallization and preliminary X-ray analysis of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae. Acta Crystallogr Sect F Struct Biol Cryst Commun 61:228-31
Wlodawer, Alexander; Li, Mi; Gustchina, Alla et al. (2004) Two inhibitor molecules bound in the active site of Pseudomonas sedolisin: a model for the bi-product complex following cleavage of a peptide substrate. Biochem Biophys Res Commun 314:638-45
Li, Tang; Yowell, Charles A; Beyer, Bret B et al. (2004) Recombinant expression and enzymatic subsite characterization of plasmepsin 4 from the four Plasmodium species infecting man. Mol Biochem Parasitol 135:101-9
Wlodawer, Alexander; Durell, Stewart R; Li, Mi et al. (2003) A model of tripeptidyl-peptidase I (CLN2), a ubiquitous and highly conserved member of the sedolisin family of serine-carboxyl peptidases. BMC Struct Biol 3:8
Wlodawer, Alexander; Li, Mi; Gustchina, Alla et al. (2003) Structural and enzymatic properties of the sedolisin family of serine-carboxyl peptidases. Acta Biochim Pol 50:81-102

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