Evidence for the existence of two soluble NAD(+) kinase isoenzymes is Euglena gracilis Z

Two soluble NAD(+) kinase isoenzymes (isoenzymes 1 and 2) from Euglena grac ilis were separated by preparative electrophoresis and characterized. They display several similar properties: both have an identical apparent molecul ar weight of 68 kDa and their activities are independent on calmodulin, ins ensitive to 2-mercaptoethanol but inhibited by p-chloromercurybenzoate, 5,5 '-dithiobis(2-nitrobenzoate) and, surprisingly, by low dithiothreitol conce ntrations, the inhibition by dithiothreitol being irreversible for isoenzym e 1 but reversible for isoenzyme 2. Nevertheless, the two isoenzymes mainly differ by their specificities towards triphosphate nucleotides and their c atalytic 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 MgATP(2)-concentrations of 0.03 mM. Isoenzyme 2 is three-fo ld more active in the presence of CTP than of ATP and operates by a sequent ial mechanism with k(M)s for NAD(+) and MgGTP(2-) of 1.03 and 0.20 mM, resp ectively. This study shows the evidence for the existence of two structural ly similar but catalytically different NAD(+) kinase isoenzymes in E. graci lis. One resembles the enzyme previously described in bacteria. The other d isplays 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. All r ights reserved.