Ca2+‐Dependent Allosteric Regulation of Nicotinamide Nucleotide Transhydrogenase from Pseudomonas aeruginosa

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Abstract
1. The addition of Ca2+ to purified nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa results in a 2–8‐fold increase in flavin fluorescence, closely resembling that induced by 2′‐AMP (adenosine 2′‐monophosphate). The response to Ca2+ is sigmoidal, with a Hill‐coefficient of 2.6. Reduction of thionicotinamide adenine dinucleotide (oxidised form) by NADH responds in a similar fashion to Ca2+ and 2′‐AMP.2. Mg2+ inhibits the Ca2+‐dependent activation in a competitive fashion, whereas no effect of Mg2+ is observed when the enzyme is activated with 2′‐AMP.3. The pH dependencies of the activations by 2′‐AMP and Ca2+ are different. The activation by Ca2+ shows little or no dependence on pH, whereas higher concentrations of 2′‐AMP are required to obtain the same activation when the pH is increased.4. The addition of both Ca2+ and 2′‐AMP causes a dramatic decrease in the concentration of Ca2+ required for half‐maximal stimulation.5. A negative cooperative effect of NADP+ has been demonstrated indirectly, i.e. addition of NADP+ increases the concentration of Ca2+ required for half‐maximal stimulation.6. These results are interpreted as indicating the existence of separate allosteric binding sites for Ca2+ and 2′‐AMP. Furthermore, Ca2+ is proposed to act as a heterotropic ligand. The results are discussed in terms of current allosteric models.