Distinctive interactions in the holoenzyme formation for two isoforms of glutamate decarboxylase.
The interactions between glutamate decarboxylase (GAD) and its cofactor pyridoxal phosphate (PLP) play a key role in the regulation of GAD activity. The enzyme has two isoforms, GAD65 and GAD67. A comparison of binding constants, rate constants, and kinetic profiles for the formation of holoenzyme (holoGAD65 and holoGAD67) revealed that the two isoforms interact distinctively with the cofactor. GAD67 exhibits a higher binding constant for PLP binding, making it more difficult to dissociate PLP from holoGAD67 than holoGAD65. Meanwhile, PLP binding occurs at a much slower rate for GAD67 than GAD65, as evidenced by lower rate constants and a slower initial rate of the holoenzyme formation. Job's plots revealed a stoichiometry of 1:1 for PLP binding to GAD65 before and after the saturation level of PLP, while 1:2 for PLP binding to GAD67 prior to the saturation of PLP and 1:1 at the saturation level of PLP. These results suggested that the two binding sites of GAD65 exhibit similar affinities for PLP. In contrast, one binding site of GAD67 exhibits a significantly higher affinity for PLP than the other binding site. Based on these findings, it was proposed that a slower PLP binding to GAD67 than GAD65 and a less ease to dissociate PLP from holoGAD67 than holoGAD65 are important underlying factors. This attributes to GAD67 being more highly saturated by PLP and GAD65 being less saturated by PLP. A larger conformation change constant for GAD67 than GAD65 supported a significant conformational change induced by the initial PLP binding to GAD67, which affects the other binding site affinity of GAD67. The present studies provided valuable insights into distinctive properties between the two isoforms of GAD.
Chen
Chang Hwei
CH
Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, NY 12201-0509, USA. chenc@wadsworth.org
Battaglioli
Gino
G
Martin
David L
DL
Hobart
Sarah A
SA
Colón
Wilfredo
W
D005453:Fluorescence; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000737:chemistry; D013816:Thermodynamics
D020035:Holoenzymes; D007527:Isoenzymes; D011732:Pyridoxal Phosphate; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2