Papain, a thiol protease from papaya latex, will be studied to determine the role of Asp-158 in catalysis and to determine the effect of Asp-158 on the interactive ionization of His-159 and Cys-25 at the active site of papain. Derivatives of papain modified at Asp-158 will be prepared and characterized with respect to their altered interactions with substrates and inhibitors. Proton NMR and potentiometric difference titrations will be used to determine alterations in the ionization behavior of His-159 and Cys-25 caused by modification of Asp-158. The interactive ionization of His-159 and Cys-25 also will be investigated to determine the effect of the ion-pair interaction on the reactivity of these residues. The nucleophilic reactivity of ammonium-thiolate ion-pairs in nonenzymic reactions will be studied to evaluate the possible catalytic advantage of the imidazolium-thiolate ion-pair at the active site of papain. The potentiometric difference titration method we have developed for determining the ionization behavior of the thiol group in papain will be used to determine the possible existence of ligand dependent ionic interactions involving Cys-beta 93 of hemoglobin. D-Serine dehydratase from E. coli will be studied to determine a) how monovalent cations effect the affinity of the enzyme for its cofactor pyridoxal 5'-phosphate, b) the involvement of a thiol group in the catalytic activity of the enzyme, and c) the intermediates in the catalytic pathway and their rates of interconversion. Studies with human fibrinogen from individuals with dysfibrinogenemia are proposed in which amino acid replacements in abnormal fibrinogens will be related to their altered functional competence especially their altered interactions with the enzymes involved in blood clot formation dissolution.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL032006-24
Application #
3343200
Study Section
Biochemistry Study Section (BIO)
Project Start
1984-04-01
Project End
1989-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
24
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Hornyak, T J; Shafer, J A (1992) Interactions of factor XIII with fibrin as substrate and cofactor. Biochemistry 31:423-9
Marceau, M; Lewis, S D; Kojiro, C L et al. (1990) Disruption of active site interactions with pyridoxal 5'-phosphate and substrates by conservative replacements in the glycine-rich loop of Escherichia coli D-serine dehydratase. J Biol Chem 265:20421-9
Hornyak, T J; Bishop, P D; Shafer, J A (1989) Alpha-thrombin-catalyzed activation of human platelet factor XIII: relationship between proteolysis and factor XIIIa activity. Biochemistry 28:7326-32
Kojiro, C L; Marceau, M; Shafer, J A (1989) Effect of potassium ion on the phosphorus-31 nuclear magnetic resonance spectrum of the pyridoxal 5'-phosphate cofactor of Escherichia coli D-serine dehydratase. Arch Biochem Biophys 268:67-73
Marceau, M; Lewis, S D; Kojiro, C L et al. (1989) Contribution of a conserved arginine near the active site of Escherichia coli D-serine dehydratase to cofactor affinity and catalytic activity. J Biol Chem 264:2753-7
Shafer, J A; Higgins, D L (1988) Human fibrinogen. Crit Rev Clin Lab Sci 26:1-41
Marceau, M; McFall, E; Lewis, S D et al. (1988) D-serine dehydratase from Escherichia coli. DNA sequence and identification of catalytically inactive glycine to aspartic acid variants. J Biol Chem 263:16926-33
Marceau, M; Lewis, S D; Shafer, J A (1988) The glycine-rich region of Escherichia coli D-serine dehydratase. Altered interactions with pyridoxal 5'-phosphate produced by substitution of aspartic acid for glycine. J Biol Chem 263:16934-41
Lewis, S D; Lorand, L; Fenton 2nd, J W et al. (1987) Catalytic competence of human alpha- and gamma-thrombin in the activation of fibrinogen and factor XIII. Biochemistry 26:7597-603
Roberts, D D; Lewis, S D; Ballou, D P et al. (1986) Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate. Biochemistry 25:5595-601

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