Enzyme kinetics determined using calorimetry: a general assay for enzyme activity?

Two techniques for determining enzyme kinetic constants using isothermal titration microcalorimetry are presented. The methods are based on the proportionality between the rate of a reaction and the thermal power (heat/time) generated. (i) An enzyme can be titrated with increasing amounts of substrate, while pseudo-first-order conditions are maintained. (ii) Following a single injection, the change in thermal power as substrate is depleted can be continuously monitored. Both methods allow highly precise kinetic characterization in a single experiment and can be used to measure enzyme inhibition. Applicability is demonstrated using a representative enzyme from each EC classification, including (i) oxidation-reduction activity of DHFR (EC 1.5.1.3); (ii) transferase activity of creatine phosphokinase (EC 2.7.3.2) and hexokinase (EC 2.7.1.1); (iii) hydrolytic activity of Helicobacter pylori urease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.21.16); (iv) lyase activity of heparinase (EC 4.1.1.7); and (v) ligase activity of pyruvate carboxylate (EC 6.4.1.1). This nondestructive method is completely general, enabling precise analysis of reactions in spectroscopically opaque solutions, using physiological substrates. Such a universal assay may have wide applicability in functional genomics.

[1]  G. Lorimer,et al.  A quantitative assessment of the role of the chaperonin proteins in protein folding in vivo , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[2]  T. Inagami.  THE MECHANISM OF THE SPECIFICITY OF TRYPSIN CATALYSIS. I. INHIBITION BY ALKYL AMMONIUM IONS. , 1964, The Journal of biological chemistry.

[3]  B. Marshall,et al.  Campylobacter pyloridis, gastritis, and peptic ulceration. , 1986, Journal of clinical pathology.

[4]  E. Freire,et al.  Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase. , 1991, Biochemistry.

[5]  W. Howe,et al.  The HIV-1 protease as enzyme and substrate: mutagenesis of autolysis sites and generation of a stable mutant with retained kinetic properties. , 1994, Biochemistry.

[6]  R. Linhardt,et al.  Purification and characterization of heparin lyases from Flavobacterium heparinum. , 1992, The Journal of biological chemistry.

[7]  G. Lorimer,et al.  Criteria for assessing the purity and quality of GroEL. , 1998, Methods in enzymology.

[8]  R. Biltonen,et al.  The application of flow microcalorimetry to the study of enzyme kinetics. , 1981, Analytical biochemistry.

[9]  C. Debouck,et al.  Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[10]  S. Colowick,et al.  MULTIPLE FORMS OF YEAST HEXOKINASE , 1961, Annals of the New York Academy of Sciences.

[11]  F. Tabita,et al.  D-ribulose 1,5-diphosphate carboxylase from Rhodospirillum rubrum. I. Levels, purification, and effects of metallic ions. , 1974, The Journal of biological chemistry.

[12]  R. Duggleby,et al.  Herbicide-resistant forms of Arabidopsis thaliana acetohydroxyacid synthase: characterization of the catalytic properties and sensitivity to inhibitors of four defined mutants. , 1998, The Biochemical journal.

[13]  E. Freire,et al.  The structural stability of the HIV-1 protease. , 1998, Journal of molecular biology.

[14]  E. Finkel.  Potential Target Found for Antimetastasis Drugs , 1999, Science.

[15]  R. Gilli,et al.  A flow microcalorimetric method for enzyme activity measurements: application to dihydrofolate reductase. , 1987, Analytical biochemistry.

[16]  G. Lorimer,et al.  Dynamics of the chaperonin ATPase cycle: implications for facilitated protein folding. , 1994, Science.

[17]  W. Farmerie,et al.  Sensitive, soluble chromogenic substrates for HIV-1 proteinase. , 1990, The Journal of biological chemistry.

[18]  A. Velázquez‐Campoy,et al.  Isothermal titration calorimetry , 1990 .

[19]  S. Chen,et al.  ATP induces large quaternary rearrangements in a cage-like chaperonin structure , 1993, Current Biology.

[20]  M. Utter,et al.  [38] Pyruvate carboxylase from chicken liver , 1969 .

[21]  Gary T. Wang,et al.  Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. , 1990, Science.

[22]  J F Brandts,et al.  Rapid measurement of binding constants and heats of binding using a new titration calorimeter. , 1989, Analytical biochemistry.

[23]  D Thirumalai,et al.  Chaperonin-facilitated protein folding: optimization of rate and yield by an iterative annealing mechanism. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Yechezkel Kashi,et al.  GroEL-mediated protein folding proceeds by multiple rounds of binding and release of nonnative forms , 1994, Cell.

[25]  G. Lorimer,et al.  Chaperonin-facilitated refolding of ribulosebisphosphate carboxylase and ATP hydrolysis by chaperonin 60 (groEL) are K+ dependent. , 1990, Biochemistry.

[26]  Zbyszek Otwinowski,et al.  The crystal structure of the bacterial chaperonln GroEL at 2.8 Å , 1994, Nature.

[27]  E. J. East,et al.  Binding of benzoamidine and protons to trypsin as measured by difference spectra. , 1968, Archives of biochemistry and biophysics.

[28]  A. Wlodawer,et al.  Structure-based inhibitors of HIV-1 protease. , 1993, Annual review of biochemistry.

[29]  F. Gillin,et al.  Properties of penicillinase from Bacillus cereus 569. , 1970, Journal of Biological Chemistry.

[30]  U. R. Desai,et al.  Specificity studies on the heparin lyases from Flavobacterium heparinum. , 1993, Biochemistry.

[31]  H. Mobley,et al.  Purification and N-terminal analysis of urease from Helicobacter pylori , 1990, Infection and immunity.

[32]  G. Lorimer,et al.  Purified chaperonin 60 (groEL) interacts with the nonnative states of a multitude of Escherichia coli proteins , 1992, Protein science : a publication of the Protein Society.

[33]  R. Linhardt,et al.  Examination of the substrate specificity of heparin and heparan sulfate lyases. , 1990, Biochemistry.

[34]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[35]  E. Toone,et al.  Calorimetric evaluation of enzyme kinetic parameters , 1993 .

[36]  I. Wadsö,et al.  Calorimetry as an analytical tool in biochemistry and biology. , 2006, Methods of biochemical analysis.

[37]  G. Watt,et al.  A microcalorimetric procedure for evaluating the kinetic parameters of enzyme-catalyzed reactions: kinetic measurements of the nitrogenase system. , 1990, Analytical biochemistry.

[38]  H. Mobley,et al.  Expression of catalytically active recombinant Helicobacter pylori urease at wild-type levels in Escherichia coli , 1993, Infection and immunity.

[39]  N. Razi,et al.  Biosynthesis of heparin/heparan sulfate. The D-glucosaminyl 3-O- sulfotransferase reaction: target and inhibitor saccharides , 1995, The Journal of Biological Chemistry.

[40]  A. Velázquez‐Campoy,et al.  Determination of the Rigorous Transfer Function of An Isothermal Titration Microcalorimeter with Peltier Compensation , 1999 .

[41]  P. B. Rowe,et al.  Dihydrofolate reductase. Studies on the activation of the bovine liver enzyme. , 1973, The Journal of biological chemistry.