Abstract

A novel universal method for measuring enzymatic activity using micro-calorimetry is applied to the HIV-1 protease (PR). Biocalorimetric assays measure the rate of heat evolution during conversion of substrate to product. Calorimetry thus gives a direct measure of the rate of an enzymatic reaction, as opposed to conventional assays where rate is calculated as a derivative of a quantity with time. Since no chromophore is necessary, the physiological substrates can be used. The activity of PR mutant (Q7K, L33I, L63I) was assayed using: 1) a standard chromogenic substrate (Arg-Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-NH2); 2) a non-chromogenic version of the same peptide (Arg-Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-NH2); and, 3) a non-chromogenic physiological substrate (Ser-Gln-Asn-Tyr-Pro-Ile-Val-NH2). The activity of PR as a function of salt concentration, pH, temperature (and ) for Km and Vmax are described. In addition, the inhibition by products pepstatin and acetyl-pepstain is described.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR004328-10
Application #
6122051
Study Section
Project Start
1997-08-05
Project End
1998-08-04
Budget Start
Budget End
Support Year
10
Fiscal Year
1997
Total Cost
Indirect Cost

  Institution

Name
Johns Hopkins University
Department
Type
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218

  Publications

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Karantza, V; Freire, E; Moudrianakis, E N (2001) Thermodynamic studies of the core histones: stability of the octamer subunits is not altered by removal of their terminal domains. Biochemistry 40:13114-23
Todd, M J; Gomez, J (2001) Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Anal Biochem 296:179-87
Griko, Y V; Remeta, D P (1999) Energetics of solvent and ligand-induced conformational changes in alpha-lactalbumin. Protein Sci 8:554-61
Chu, V; Freitag, S; Le Trong, I et al. (1998) Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin-biotin system. Protein Sci 7:848-59
Luque, I; Freire, E (1998) Structure-based prediction of binding affinities and molecular design of peptide ligands. Methods Enzymol 295:100-27
Luque, I; Gomez, J; Semo, N et al. (1998) Structure-based thermodynamic design of peptide ligands: application to peptide inhibitors of the aspartic protease endothiapepsin. Proteins 30:74-85
Gomez, J; Semo, N; Freire, E (1998) Structural thermodynamic study of the binding of renin inhibitors to endothiapepsin. Adv Exp Med Biol 436:325-8
Koder, R L; Miller, A F (1998) Overexpression, isotopic labeling, and spectral characterization of Enterobacter cloacae nitroreductase. Protein Expr Purif 13:53-60
Freire, E (1998) Statistical thermodynamic linkage between conformational and binding equilibria. Adv Protein Chem 51:255-79

Showing the most recent 10 out of 86 publications