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Identifier

000375001

Title

Γλουταμική αφυδρογονάση ειδική για το νευρικό σύστημα : επίδραση ρυθμιστικών μεταλλάξεων στη βασική δραστηριότητα και τη δομή του ενζύμου

Alternative Title

Nervous-system specific glutamate dehydrogenase:

Author

Καναβούρας, Κωνσταντίνος Ε

Thesis advisor

Κυριακή, Θερμού

Reviewer

Πλαιτάκης, Ανδρέας
Κοκκινίδης, Μιχαήλ
Καρδάσης, Δημήτριος
Καραγωγέως, Δόμνα
Σπανάκη, Κλεάνθη
Χαραλαμπόπουλος, Ιωάννης

Abstract

Glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism and subject to complex allosteric regulation, exists in humans in two isoforms: the housekeeping hGDH1, encoded by the GLUD1 gene, and hGDH2, which is expressed mainly in brain and testis and encoded by GLUD2, a gene that emerged through retroposition ~20 million years ago. Despite 97% sequence homology, with only 15 aminoacid differences between hGDH1 and hGDH2, the two isoenzymes differ markedly in their properties: hGDH2 displays a substantially lower basal activity in absence of activators, increased resistance to GTP inhibition, and decreased thermostability. In this work we explore structure-function relationships of the two isoenzymes through mutagenesis and functional studies on wild-type and mutant hGDHs. We show that basal activity of hGDH2 in vitro is dependent on enzyme concentration, temperature and buffer composition. Furthermore, we show that the aminoacid differences R443S and G456A in the allosteric region of the enzyme, shown previously to diminish basal activity and to increase GTP-resistance, respectively, cannot reproduce the unique properties of hGDH2: the hGDH1- R443S/G456A double mutant has much lower basal activity and heat resistance than hGDH2, and is not activated by leucine. A chimeric enzyme, featuring all 4 aminoacid differences in the allosteric region (hGDH1-M415L/R443S/G456A/R470H), can also not reproduce the properties of hGDH2. The S174N aminoacid change, however, introduced into the R443S/G456A double mutant, makes the resulting enzyme sensitive to leucine activation and increases its basal activity and heat stability. Thus, aminoacid changes outside the allosteric region are crucial for the unique properties of hGDH2. In addition, we show that basal activity, heat stability and resistance to inhibition by estrogens are directly related, interlinked to each other properties of hGDHs. Finally, we show that a polymorphism in hGDH2 (S445A), that has been associated to accelerated onset of Parkinson’s disease, probably exerts its effect by increasing hGDH2’s basal activity and does not affect GTP inhibition. Mutations in the same region of hGDH1 (Q441R, S445L, S448P, K450E, H454Y), which cause the hyperinsulinism-hyperammonemia syndrome through increasing hGDH1’s resistance to GTP, do not have a similar effect in GTP-regulation when introduced to hGDH2, but can instead cause alterations in basal activity, depending on their exact location.

Language

Greek

Subject

Biochemistry

Glud1

Glud2

Glutamate

Glutamate dehydrogenase

Parkinsons disease

Structure-function relationship

Γλουταμική αφυδρογονάση

Γλουταμικό