Single Amino Acid Substitution (G456A) in the Vicinity of the GTP Binding Domain of Human Housekeeping Glutamate Dehydrogenase Markedly Attenuates GTP Inhibition and Abolishes the Cooperative Behavior of the Enzyme

Ioannis Zaganas,Andreas Plaitakis

doi:10.1074/jbc.m200022200

Abstract

Human glutamate dehydrogenase (GDH) exists in two isoforms encoded by the GLUD1 and GLUD2 genes, respectively. Although the two enzymes share in their mature form all but 15 of their 505 amino acids, they differ markedly in their allosteric regulation. To identify the structural basis for these allosteric characteristics, we performed site-directed mutagenesis on the human GLUD1 gene at sites that differ from the GLUD2 gene using a cloned GLUD1 cDNA. Results showed that substitution of Ala for Gly-456, but not substitution of His for Arg-470 or Ser for Asn-498, renders the enzyme markedly resistant to GTP inhibition (IC(50) = 2.80 microm) as compared with the wild type GLUD1-derived GDH (IC(50) = 0.19 microm). The G456A mutation abolished the cooperative behavior of the enzyme, as revealed by the GTP inhibitory curves. The catalytic and kinetic properties of the G456A mutant and its activation by ADP were comparable with those of the wild type GDH. Gly-456 lies in a very tightly packed region of the GDH molecule, and its replacement by Ala may lead to steric clashes with neighboring amino acids. These, in turn, may affect the conformational state of the protein that is essential for the allosteric regulation of the enzyme by GTP.

Highlights

Glutamate dehydrogenase (GDH)1 (E.C.1.4.1.3) catalyzes the reversible oxidative deamination of glutamate to ␣-ketoglutarate using NAD(H) or NADP(H) as cofactors [1]
Three residues (Gly-456, Arg-470, and Ser-498) that are different in the GLUD1 as compared with the GLUD2 gene were selected for these studies (Fig. 1)
Results showed that replacement of Gly by Ala at site 456 markedly attenuates GTP inhibition and abolishes the cooperative behavior of the enzyme without affecting its kinetic properties

Summary

Introduction

Glutamate dehydrogenase (GDH) (E.C.1.4.1.3) catalyzes the reversible oxidative deamination of glutamate to ␣-ketoglutarate using NAD(H) or NADP(H) as cofactors [1]. The two GDH isoenzymes are highly homologous (showing a 97% amino acid identity), they differ markedly in their regulatory properties [8, 10]. We selected three such residues (Gly-456, Arg-470, and Asn-498) located in the C-terminal region, which is thought to be part of the regulatory domain of mammalian GDH [11]. Using site-directed mutagenesis, we created three GLUD1 mutants, each containing one of these amino substitutions In each of these sites, the amino acid residue present in the GLUD2 GDH replaced the corresponding amino acid of the GLUD1 enzyme. Results showed that substitution of Ala for Gly at position 456 (but not substitution of His for Arg-470 or Ser for Asn-498) of the GLUD1 GDH markedly attenuated GTP inhibition and abolished the cooperative behavior of the enzyme. The structural, functional, and evolutionary implications of these findings are discussed

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Conclusion