title: Kinetic behavior of the Arabidopsis thaliana leaf formate dehydrogenase is thermally sensitive.
abstract: Two previous kinetic studies on the Arabidopsis thaliana leaf NAD-dependent formate dehydrogenase (EC 1.2.1.2) have demonstrated two very different sets of Km values for the formate and NAD+ substrates. We examined the kinetics of the enzyme partially purified from a leaf extract by gel-filtration desalting and chromatography on DEAE-cellulose, as well as by isolation of a mitochondria-enriched fraction obtained by differential centrifugation. Both of these methods produce a formate dehydrogenase enzyme with the higher Km values of approximately 10 mmol/L formate and 75 mumol/L NAD+. The kinetic properties of the Arabidopsis formate dehydrogenase expressed to high levels in transgenic tobacco plants were also those of the high Km form. The high Km form of the enzyme converted to a low Km form by heating for 5 minutes at 60 degrees C. An Arrhenius plot of the activity during the heating process was linear, indicating that the heating did not cause alterations in either the active site or the thermal dependence of the catalytic reaction. We conclude that the native form of the formate dehydrogenase probably resembles the form with the higher Km values. Heating seemingly converts this native enzyme to the molten globule state and cooling results in formation of a non-native structure with altered kinetic properties.
authors.affiliation: Department of Biochemistry, University of Nebraska, Lincoln, NE 68588-0664, USA.
authors.forename: Renee D
authors.initials: RD
authors.lastname: Baack
authors.forename: John
authors.initials: J
authors.lastname: Markwell
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Herman
authors.forename: John C
authors.initials: JC
authors.lastname: Osterman
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D005560:Formate Dehydrogenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D018515:Plant Leaves / Q000201:enzymology*; D030821:Plants, Genetically Modified; D014026:Nicotiana
chemical_list: C037212:formate dehydrogenase (NADP+); D005560:Formate Dehydrogenases