Amino acid changes within antenna helix are responsible for different regulatory preferences of human glutamate dehydrogenase isozymes.
Human glutamate dehydrogenase (hGDH) exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. Because they differ in only 16 of their 505 amino acids, the regulatory preferences must arise from amino acid residues that are not common between hGDH1 and hGDH2. To our knowledge none of the mutagenesis studies on the hGDH isozymes to date have identified the amino acid residues fully responsible for the different regulatory preferences between hGDH1 and hGDH2. In this study we constructed hGDH1(hGDH2(390-448))hGDH1 (amino acid segment 390-448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1(390-448))hGDH2 (amino acid segment 390-448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments in hGDH1 and hGDH2. The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2 and are located in the C-terminal 48-residue "antenna" helix, which is thought to be part of the regulatory domain of mammalian GDHs. Functional analyses revealed that the doubly mutated chimeric enzymes almost completely acquired most of the different regulatory preferences between hGDH1 and hGDH2 for electrophoretic mobility, heat-stability, ADP activation, palmitoyl-CoA inhibition, and l-leucine activation, except for GTP inhibition. Our results indicate that substitutions of the residues in the antenna region may be important evolutionary changes that led to the adaptation of hGDH2 to the unique metabolic needs of the nerve tissue.
Choi
Myung-Min
MM
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 138-736, Korea.
Kim
Eun-A
EA
Yang
Seung-Ju
SJ
Choi
Soo Young
SY
Cho
Sung-Woo
SW
Huh
Jae-Wan
JW
D000494:Allosteric Regulation / Q000235:genetics; D019943:Amino Acid Substitution; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability / Q000235:genetics; D019143:Evolution, Molecular; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009928:Organ Specificity / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D005969:Glutamate Dehydrogenase