title: A transient-kinetic study of the nitrogenase of Klebsiella pneumoniae by stopped-flow calorimetry. Comparison with the myosin ATPase.
abstract: The pre-steady-state kinetics of MgATP hydrolysis by nitrogenase from Klebsiella pneumoniae were studied by stopped-flow calorimetry at 6 degrees C and at pH 7.0. An endothermic reaction (delta Hobs. = +36 kJ.mol of ATP-1; kobs. = 9.4 s-1) in which 0.5 proton.mol of ATP-1 was released, has been assigned to the on-enzyme cleavage of MgATP to yield bound MgADP + Pi. The assignment is based on the similarity of these parameters to those of the corresponding reaction that occurs with rabbit muscle myosin subfragment-1 (delta Hobs. = +32 kJ.mol of ATP-1; kobs. = 7.1 s-1; 0.2 proton released.mol of ATP-1) [Millar, Howarth & Gutfreund (1987) Biochem. J. 248, 683-690]. MgATP-dependent electron transfer from the nitrogenase Fe-protein to the MoFe-protein was monitored by stopped-flow spectrophotometry at 430 nm and occurred with kobs. value of 3.0 s-1 at 6 degrees C. Thus, under these conditions, hydrolysis of MgATP precedes electron transfer within the protein complex. Evidence is presented that suggests that MgATP cleavage and subsequent electron transfer are reversible at 6 degrees C with an overall equilibrium constant close to unity, but that, at 23 degrees C, the reactions are essentially irreversible, with an overall equilibrium constant greater than or equal to 10.
authors.affiliation: AFRC Institute of Plant Science Research, University of Sussex, Brighton, U.K.
authors.forename: R N
authors.initials: RN
authors.lastname: Thorneley
authors.forename: G
authors.initials: G
authors.lastname: Ashby
authors.forename: J V
authors.initials: JV
authors.lastname: Howarth
authors.forename: N C
authors.initials: NC
authors.lastname: Millar
authors.forename: H
authors.initials: H
authors.lastname: Gutfreund
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002151:Calorimetry; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008954:Models, Biological; D009591:Nitrogenase / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000255:Adenosine Triphosphate; D009591:Nitrogenase