Two soluble NAD+ kinase isoenzymes (isoenzymes 1 and 2) from Euglena gracilis were separated by preparative electrophoresis and characterized. They display several similar properties: both have an identical apparent molecular weight of 68 kDa and their activities are independent on calmodulin, insensitive to 2-mercaptoethanol but inhibited by p- chloromercurybenzoate, 5,5'-dithiobis(2-nitrobenzoate) and, surprisingly, by low dithiothreitol concentrations, the inhibition by dithiothreitol being irreversible for isoenzyme 1 but reversible for isoenzyme 2. Nevertheless, the two isoenzymes mainly differ by their specificities towards triphosphate nucleotides and their catalytic mechanisms. Isoenzyme 1 is as active in the presence of ATP as of GTP and acts by a ping-pong mechanism with a k(M) for NAD+ of 0.26 mM and a k(M) for low MgATP2-concentrations of 0.03 mM. Isoenzyme 2 is three-fold more active in the presence of GTP than of ATP and operates by a sequential mechanism with k(M)s for NAD+ and MgGTP2- of 1.03 and 0.20 mM, respectively. This study shows the evidence for the existence of two structurally similar but catalytically different NAD+ kinase isoenzymes in E. gracilis. One resembles the enzyme previously described in bacteria. The other displays a catalytic mechanism identical to that of NAD+ kinase from other organisms but remains unique among all the NAD+ kinases studied to-date regarding its specificity towards GTP. (C) 2000 Elsevier Science Ltd.
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