Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes¹

Abstract

Molecular biological studies con¢rmed that two glutamate dehydrogenase isozymes (hGDH1 and hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat-stable and expressed widely, whereas hGDH2 is heat-labile and speci¢c for neural and testicular tissues. A selective de¢-ciency of hGDH2 has been reported in patients with spinocerebellar ataxia. We have identi¢ed an amino acid residue involved in the di¡erent thermal stability of human GDH isozymes. At 45 ‡C (pH 7.0), heat inactivation proceeded faster for hGDH2 (half life = 45 min) than for hGDH1 (half-life = 310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L-Leu. Virtually most of the enzyme activity remained up to 100 min at 45 ‡C after treatment with ADP and L-Leu in combination. In contrast to ADP and L-Leu, the thermal stabilities of the hGDH isozymes were not a¡ected by addition of substrates or coenzymes. In human GDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2. Replacement of Ser by Arg at the 443 site by cassette mutagenesis abolished the heat lability of hGDH2 with a similar half-life of hGDH1. The mutagenesis at several other sites (L415M, A456G, and H470R) having di¡erences in amino acid sequence between the two GDH isozymes did not show any change in the thermal stability. These results suggest that the Ser443 residue plays an important role in the di¡erent thermal stability of human GDH isozymes.

Regulation of glutamate level in rat brain through activation of glutamate dehydrogenase by Corydalis ternata

“…It also indicates that the different sensitivities to alkalized extracts in the presence of high concentration of ADP may due to differences in the regulatory properties between hGDH isozymes by ADP. These observations are consistent with the previous reports that there are at least two different GDH activities differing in their relative thermal stability and allosteric regulation characteristics (Mavrothalassitis et al, 1988;Shashidharan et al, 1994;Cho et al, 2001;Yang et al, 2004). Since physiological ADP levels can vary from 0.05 to 1.0 mM depending on the rate of oxidative phosphorylation, our results suggest a possibility that hGDH1 and hGDH2 are differently regulated in vivo by the actions of alkalized extracts depending on the physiological concentrations of ADP.…”

“…Human GDH genes (pHGDH1 and pHGDH2) have been chemically synthesized, expressed in E. coli as soluble proteins, and homogeneously purified by the same methods developed in our laboratory as described elsewhere Lee et al, 2001;Yoon et al, 2002a;Yang et al, 2004). Y187M mutant at the ADP-binding site of the hGDH isozymes was constructed and homogeneously purified by the same methods developed in our laboratory as described elsewhere Yoon et al, 2002b).…”

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

“…Although this enzyme does not exhibit allosteric regulation in plants, bacteria, or fungi, its activity is tightly controlled by a number of compounds in mammals (1)(2)(3)(4)(5)(6). All mammalian GDHs are homohexameric, exhibiting 32 symmetry, and the first 200 residues form the core "glutamate binding" domain.…”

“…Reciprocally interchanging amino acids that are different between hGDH isozymes within the regulatory domain may reveal the residues important for preference in hGDH isozymes. Recent studies of structure-function relationships using site-directed mutagenesis of hGDH1 at single sites differing from hGDH2 showed that the R443S and the G456A change reproduced some but not all of the properties of hGDH2 (3)(4)(5)17). In addition, several other residues differing between hGDH isozymes also have been examined by many investigators (4,17).…”

“…Recent studies of structure-function relationships using site-directed mutagenesis of hGDH1 at single sites differing from hGDH2 showed that the R443S and the G456A change reproduced some but not all of the properties of hGDH2 (3)(4)(5)17). In addition, several other residues differing between hGDH isozymes also have been examined by many investigators (4,17). Very recently, Kanavouras et al (6) created a double hGDH1 mutant differing from hGDH2 and showed that the double-mutated enzyme with R443S and G456A in the same polypeptide chain did not acquire all the characteristics of the wild-type hGDH2.…”