ATP's impact on the conformation and holoenzyme formation in relation to the regulation of brain glutamate decarboxylase.
To investigate ATP as a potential factor in the regulation of brain glutamate decarboxylase (GAD), the impact of ATP on the enzyme conformation and holoenzyme formation was investigated. ATP at 100 microM quenches fluorescence emission intensity of the holoenzyme of GAD (holoGAD) by 18% after a correction for the inner filter effect and enhances fluorescence steady-state polarization from 0.158 to 0. 183 when excited at 280 or 295 nm. These findings suggest that ATP moderately changes the microenvironment of one or more tryptophan or tyrosine residues in holoGAD and alters these residues from a more mobile state to a less mobile one. A moderate ATP-induced conformational change in holoGAD is also supported by the observations that ATP increases the thermal denaturation temperature of holoGAD by 2 degrees C, as derived from temperature-dependent fluorescence spectra, and decreases the alpha-helical content of holoGAD by 8-10%, as determined by circular dichroism. Moreover, ATP does not affect the keto-enol tautomerization of holoGAD and has little or no direct effect on its activity, implying that the ATP interacting domain in holoGAD is not at the active site. Kinetics studies, as demonstrated by stopped-flow fluorescence and UV/visible spectroscopy, demonstrate that formation of holoGAD involves two steps: a fast reaction forming an apoGAD-cofactor intermediate complex, followed by a slow reaction involving the conformational change in the intermediate complex. ATP reduces the rate constant of the fast step to one-third and decreases the rate of the slow step and the intermediate complex formation constant to 60% of their original values. The present data suggest that ATP may regulate the interconversion between apoGAD and holoGAD by interacting with apoGAD rather than holoGAD. By slowing down the rate of intermediate complex formation, ATP reduces the amount of holoGAD formed.
Chen
C H
CH
Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509, USA.
Colón
W
W
Myer
Y P
YP
Martin
D L
DL
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002942:Circular Dichroism; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000378:metabolism*; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D001051:Apoenzymes; D020035:Holoenzymes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D005968:Glutamate Decarboxylase