Evidence for the existence of two soluble NAD(+) kinase isoenzymes in Euglena gracilis Z.
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 MgATP(2-)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 MgGTP(2-) 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.
GBBMV-EA917, UFR Sciences, 2 Boulevard Lavoisier, 49045, Angers, France.
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Montrichard
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005056:Euglena gracilis / Q000166:cytology / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D002147:Calmodulin; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D013438:Sulfhydryl Compounds; D009243:NAD; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase; D002118:Calcium