[en] A new microcalorimetric method for recording the kinetic parameters k(cat)/K-m and K-i of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic a-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic a-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic a-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site. (c) 2006 Elsevier Ltd. All rights reserved.
Junge K., Eicken H., and Deming J.W. Bacterial activity at -2 to -20 degrees C in Arctic wintertime sea ice. Appl. Environ. Microbiol. 70 (2004) 550-557
Feller G., and Gerday C. Psychrophilic enzymes: hot topics in cold adaptation. Nature Rev. Microbiol. 1 (2003) 200-208
Margesin R., Feller G., Gerday C., and Russell N.J. Cold-adapted microorganisms: adaptation strategies and biotechnological potential. In: Bitton G. (Ed). Encyclopedia of Environmental Microbiology vol. 2 (2002), Wiley, New York 871-885
Cavicchioli R., Thomas T., and Curmi P.M.G. Cold stress response in archaea. Extremophiles 4 (2000) 321-331
Lim J., Thomas T., and Cavicchioli R. Low temperature regulated DEAD-box RNA helicase from the Antarctic archaeon, Methanococcoides burtonii. J. Mol. Biol. 297 (2000) 553-567
Medigue C., Krin E., Pascal G., Barbe V., Bernsel A., Bertin P.N., et al. Coping with cold: the genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. Genome Res. 15 (2005) 1325-1335
Saunders N.F., Thomas T., Curmi P.M., Mattick J.S., Kuczek E., Slade R., et al. Mechanisms of thermal adaptation revealed from the genomes of the Antarctic Archaea Methanogenium frigidum and Methanococcoides burtonii. Genome Res. 13 (2003) 1580-1588
Rabus R., Ruepp A., Frickey T., Rattei T., Fartmann B., Stark M., et al. The genome of Desulfotalea psychrophila, a sulfate-reducing bacterium from permanently cold Arctic sediments. Environ. Microbiol. 6 (2004) 887-902
Methe B.A., Nelson K.E., Deming J.W., Momen B., Melamud E., Zhang X., et al. The psychrophilic lifestyle as revealed by the genome sequence of Colwellia psychrerythraea 34H through genomic and proteomic analyses. Proc. Natl Acad. Sci. USA 102 (2005) 10913-10918
D'Amico S., Claverie P., Collins T., Georlette D., Gratia E., Hoyoux A., et al. Molecular basis of cold adaptation. Phil. Trans. Roy. Soc. ser. B 357 (2002) 917-925
Feller G. Molecular adaptations to cold in psychrophilic enzymes. Cell. Mol. Life Sci. 60 (2003) 648-662
Georlette D., Blaise V., Collins T., D'Amico S., Gratia E., Hoyoux A., et al. Some like it cold: biocatalysis at low temperatures. FEMS Microbiol. Rev. 28 (2004) 25-42
Wintrode P.L., and Arnold F.H. Temperature adaptation of enzymes: lessons from laboratory evolution. Advan. Protein Chem. 55 (2000) 161-225
Tehei M., Franzetti B., Madern D., Ginzburg M., Ginzburg B.Z., Giudici-Orticoni M.T., et al. Adaptation to extreme environments: macromolecular dynamics in bacteria compared in vivo by neutron scattering. EMBO Rep. 5 (2004) 66-70
D'Amico S., Gerday C., and Feller G. Structural similarities and evolutionary relationships in chloride-dependent alpha-amylases. Gene 253 (2000) 95-105
Da Lage J.L., Feller G., and Janecek S. Horizontal gene transfer from Eukarya to bacteria and domain shuffling: the alpha-amylase model. Cell. Mol. Life Sci. 61 (2004) 97-109
Qian M., Haser R., and Payan F. Structure and molecular model refinement of pig pancreatic alpha-amylase at 2.1 Å resolution. J. Mol. Biol. 231 (1993) 785-799
Aghajari N., Feller G., Gerday C., and Haser R. Crystal structures of the psychrophilic α-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor. Protein Sci. 7 (1998) 564-572
Aghajari N., Feller G., Gerday C., and Haser R. Structures of the psychrophilic Alteromonas haloplanctis α-amylase give insights into cold adaptation at a molecular level. Structure 6 (1998) 1503-1516
Qian M., Haser R., Buisson G., Duee E., and Payan F. The active center of a mammalian alpha-amylase. Structure of the complex of a pancreatic alpha-amylase with a carbohydrate inhibitor refined to 2.2-Å resolution. Biochemistry 33 (1994) 6284-6294
Aghajari N., Roth M., and Haser R. Crystallographic evidence of a transglycosylation reaction: ternary complexes of a psychrophilic alpha-amylase. Biochemistry 41 (2002) 4273-4280
Feller G., d'Amico D., and Gerday C. Thermodynamic stability of a cold-active α-amylase from the Antarctic bacterium Alteromonas haloplanctis. Biochemistry 38 (1999) 4613-4619
Feller G., Payan F., Theys F., Qian M., Haser R., and Gerday C. Stability and structural analysis of α-amylase from the Antarctic psychrophile Alteromonas haloplanctis A23. Eur. J. Biochem. 222 (1994) 441-447
D'Amico S., Gerday C., and Feller G. Structural determinants of cold adaptation and stability in a large protein. J. Biol. Chem. 276 (2001) 25791-25796
D'Amico S., Gerday C., and Feller G. Temperature adaptation of proteins: engineering mesophilic-like activity and stability in a cold-adapted alpha-amylase. J. Mol. Biol. 332 (2003) 981-988
D'Amico S., Marx J.C., Gerday C., and Feller G. Activity-stability relationships in extremophilic enzymes. J. Biol. Chem. 278 (2003) 7891-7896
Feller G., Bussy O., Houssier C., and Gerday C. Structural and functional aspects of chloride binding to Alteromonas haloplanctis alpha-amylase. J. Biol. Chem. 271 (1996) 23836-23841
Aghajari N., Feller G., Gerday C., and Haser R. Structural basis of alpha-amylase activation by chloride. Protein Sci. 11 (2002) 1435-1441
Lonhienne T., Baise E., Feller G., Bouriotis V., and Gerday C. Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases. Biochim. Biophys. Acta 1545 (2001) 349-356
Todd M.J., and Gomez J. Enzyme kinetics determined using calorimetry: a general assay for enzyme activity?. Anal. Biochem. 296 (2001) 179-187
Goldberg R.N. Thermodynamics of enzyme-catalyzed reactions: Part 6-1999 update. J. Phys. Chem. Ref. Data 28 (1999) 931-965
Goldberg R.N., Bell D., Tewari Y.B., and McLaughlin M.A. Thermodynamics of hydrolysis of oligosaccharides. Biophys. Chem. 40 (1991) 69-76
Rauscher E., Neumann U., Schaich E., von Bulow S., and Wahlefeld A.W. Optimized conditions for determining activity concentration of alpha-amylase in serum, with 1,4-alpha-d-4-nitrophenylmaltoheptaoside as substrate. Clin. Chem. 31 (1985) 14-19
Seigner C., Prodanov E., and Marchis-Mouren G. On porcine pancreatic alpha-amylase action: kinetic evidence for the binding of two maltooligosaccharide molecules (maltose, maltotriose and o-nitrophenylmaltoside) by inhibition studies. Correlation with the five-subsite energy profile. Eur. J. Biochem. 148 (1985) 161-168
Turnbull W.B., and Daranas A.H. On the value of c: can low affinity systems be studied by isothermal titration calorimetry?. J. Am. Chem. Soc. 125 (2003) 14859-14866
Fukada H., and Takahashi K. Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride. Proteins: Struct. Funct. Genet. 33 (1998) 159-166
Feller G., D'Amico S., Benotmane A.M., Joly F., Van Beeumen J., and Gerday C. Characterization of the C-terminal propeptide involved in bacterial wall spanning of alpha-amylase from the psychrophile Alteromonas haloplanctis. J. Biol. Chem. 273 (1998) 12109-12115
Collins T., Meuwis M.A., Gerday C., and Feller G. Activity, stability and flexibility in glycosidases adapted to extreme thermal environments. J. Mol. Biol. 328 (2003) 419-428
Georlette D., Damien B., Blaise V., Depiereux E., Uversky V.N., Gerday C., and Feller G. Structural and functional adaptations to extreme temperatures in psychrophilic, mesophilic, and thermophilic DNA ligases. J. Biol. Chem. 278 (2003) 37015-37023
Siddiqui K.S., Feller G., D'Amico S., Gerday C., Giaquinto L., and Cavicchioli R. The active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase. J. Bacteriol. 187 (2005) 6197-6205
Fields P.A., and Somero G.N. Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A(4) orthologs of Antarctic notothenioid fishes. Proc. Natl Acad. Sci. USA 95 (1998) 11476-11481
Xu Y., Feller G., Gerday C., and Glansdorff N. Metabolic enzymes from psychrophilic bacteria: challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi. J. Bacteriol. 185 (2003) 2161-2168
Fersht A. Enzyme Structure and Mechanism (1985), Freeman and Co., New York
Wiegand G., Epp O., and Huber R. The crystal structure of porcine pancreatic alpha-amylase in complex with the microbial inhibitor Tendamistat. J. Mol. Biol. 247 (1995) 99-110
