NADP-Isocitrate Dehydrogenase fromPseudomonas nautica: Kinetic Constant Determination and Carbon Limitation Effects on the Pool of Intracellular Substrates

Abstract

ABSTRACTVariations of intracellular concentrations of isocitrate and NADP+were measured throughout all growth phases of the marine bacteriumPseudomonas nautica. The intracellular isocitrate concentration tracked the intracellular protein concentration throughout all phases of growth. It rapidly increased in early exponential phase to a maximum and fell to nearly zero in parallel with pyruvate exhaustion in the culture medium. The intracellular NADP+and protein concentrations increased in parallel during the exponential phase but were poorly correlated. Even after carbon exhaustion, the intracellular NADP+concentration stayed high, as did protein levels. The results demonstrated that the intracellular isocitrate concentration, but not the intracellular NADP+concentration, was affected by the carbon availability in the culture. They also suggest that, because of its variability, isocitrate, but not NADP+, plays the larger role in the control of the respiratory CO2production rate (RCO2). From initial rate studies, bisubstrate Michaelis constants and the dissociation constant were determined for NADP+-specific isocitrate dehydrogenase (IDH) fromP. nautica. These studies support the hypothesis that the mechanism of IDH’s activity involves the ordered addition of the substrates,d-isocitrate and NADP+. Furthermore, the results support the use of a bisubstrate enzyme kinetic equation to modelRCO2inP. nautica.