NADP-isocitrate dehydrogenase from Pseudomonas nautica: Kinetic constant determination and carbon limitation effects on the pool of intracellular substrates

Variations of intracellular concentrations of isocitrate and NADP(+) were m easured throughout all growth phases of the marine bacterium Pseudomonas na utica. The intracellular isocitrate concentration tracked the intracellular protein concentration throughout all phases of growth. It rapidly increase d in early exponential phase to a maximum and fell to nearly zero in parall el with pyruvate exhaustion in the culture medium. The intracellular NADP() and protein concentrations increased in parallel during the exponential p hase but were poorly correlated. Even after carbon exhaustion, the intracel lular 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 availabilit y in the culture, They also suggest that, because of its variability, isoci trate, hut not NADP(+), plays the larger role in the control of the respira tory CO2 production rate (R-CO2). From initial rate studies, bisubstrate Mi chaelis constants and the dissociation constant were determined for NADP(+) -speeific isocitrate dehydrogenase (IDH) from P, nautica. These studies sup port the hypothesis that the mechanism of IDH's activity involves the order ed addition of the substrates, D-isocitrate and NADP(+). Furthermore, the r esults support the use of a bisubstrate enzyme kinetic equation to model R- CO2 in P. nautica.