Amino Acid Changes within Antenna Helix Are Responsible for Different Regulatory Preferences of Human Glutamate Dehydrogenase Isozymes

Abstract

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.

Novel isoenzyme of 2‐oxoglutarate dehydrogenase is identified in brain, but not in heart

“…Because of this, the distribution of 2‐oxoglutarate between irreversible degradation by OGDHC and transformation to glutamate must be subject to more diverse regulation in the brain. OGDH isoenzymes with different affinities to 2‐oxoglutarate extend regulatory means to control the glutamate/2‐oxoglutarate ratio, which is governed by the brain‐specific isoenzymes of glutamate dehydrogenase and isoforms of mitochondrial glutamate carrier [41].…”

Roles of cysteine residues in the inhibition of human glutamate dehydrogenase by palmitoyl-CoA

“…To make hGDH2 mutant proteins (C59A, C93A, C119A, C201A, C274A, and C323A), amino acid substitutions at six different Cys sites were constructed by cassette mutagenesis of synthetic hGDH2 gene (pHGDH2) as described elsewhere (15,26,34). Plasmid DNA was digested with restriction enzymes to remove the flanking fragment that encodes target amino acid.…”

“…Cell pellets were suspended in 100 ml of 100 mM Tris-HCl, pH 7.4/1 mM EDTA/5 mM dithiothreitol and lysed with a sonicator. Cellular debris was removed by centrifugation and the mutant proteins were purified by ADP-Sepharose column followed by FPLC Resource-Q column as described elsewhere (15,26,34). The purified mutant proteins were analyzed by SDS-PAGE and the western blot analysis as reported elsewhere (37,38).…”

“…Glutamate dehydrogenase is inhibited by palmitoyl-CoA and the inhibition is further enhanced by α-ketoglutarate and malate (13-15). Palmitoyl-CoA inhibition is the most primitive form of allosteric inhibition and appears to also be dependent upon other allosteric regulators (14,16).…”

Υποκυτταρικός Προορισμός Και Μηχανισμοί Μιτοχονδριακής Στόχευσης Των Ανθρώπινων Μορφών Της Γλουταμικής Αφυδρογονάσης

“…In mammals, the highest GDH activity is found in the liver where it localizes to all hepatocytes. In this organ, GDH is one of the most abundant proteins [1% (w/w) of proteins present in whole liver homogenate], constituting ∼10% of mitochondrial matrix proteins [23]. Krebs originally suggested that these huge enzyme levels are needed for keeping its reactants in equilibrium [24].…”