A Pro/Ser substitution in nucleoside diphosphate kinase of Drosophila melanogaster (mutation killer of prune) affects stability but not catalytic efficiency of the enzyme.
Nucleoside diphosphate kinase of Drosophila, recently identified as the product of the awd gene, is essential for larval development. The conditional lethal mutation Killer of prune maps to the same gene. We purified the nucleoside diphosphate kinases from wild-type and mutant larvae by a simple procedure involving affinity chromatography on blue Sepharose. Both proteins are purified as hexamers in their native state. The mutant protein, which carries a serine instead of proline at position 97, has structural properties and catalytic efficiency that are very similar to the wild-type protein. However, the mutant protein has a much lower stability to denaturation by heat and urea. Following dilution of urea with buffer the urea-denaturated mutant nucleoside diphosphate kinase accumulates as folded monomers and cannot recover its quaternary structure and enzymatic activity. In contrast, the wild-type enzyme recovers hexameric structure and activity. This suggests that the mutation affects the folding/assembly pathway without affecting the function of the mature protein once folded and assembled into the mature hexameric structure.
Lascu
I
I
Unité de Biochimie Cellulaire (URA 1129), Institut Pasteur, Paris, France.
Chaffotte
A
A
Limbourg-Bouchon
B
B
Véron
M
M
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002384:Catalysis; D002850:Chromatography, Gel; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D007301:Insect Hormones / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007525:Isoelectric Focusing; D007700:Kinetics; D009154:Mutation; D009701:Nucleoside-Diphosphate Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D011489:Protein Denaturation; D012694:Serine / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D000596:Amino Acids; D029721:Drosophila Proteins; D007301:Insect Hormones; D012694:Serine; D014508:Urea; D011392:Proline; D009701:Nucleoside-Diphosphate Kinase; C061642:awd protein, Drosophila