Characterization of NAD-dependent mannitol dehydrogenase from celery as affected by ions, chelators, reducing agents and metabolites
Stoop JMH., Williamson JD., Conkling MA., MacKay JJ., Pharr DM.
NAD-dependent mannitol dehydrogenase (MTD) from celery (Apium graveolens L. var. dulce (Mill.) Pers.) provides the initial step by which mannitol is committed to central metabolism and plays a critical role in regulating mannitol concentration in the plant. The pH optimum for mannitol oxidation occurs at pH 9.5 whereas the optimum for mannose reduction occurs at pH 6.5. Michaelis-Menten kinetics were exhibited for mannitol and NAD with Km values of 64 and 0.14 mM, respectively at pH 9.5. The K(m) for mannose and NADH were 745 mM and 1.27 μM, respectively at pH 6.5. The high K(m) for mannose is consistent with a reaction in situ favoring mannitol oxidation rather than mannose reduction. The observed down-regulation of MTD in salt stressed celery is not due to a direct inhibition by NaC1 or macronutrients. Inhibition by the chelator 1,10-phenanthroline suggests that zinc is required for MTD activity. Reducing agents DTT, DTE and β-mercaptoethanol inactivated MTD reversibly. At pH 7.0, ADP and to a lesser extend AMP and ATP were competitive inhibitors, with respect to NAD, having apparent K(i)'s of 0.24, 0.64 and 1.10 mM, respectively.