Probing the active site of the reconstituted aspartate/glutamate carrier from bovine heart mitochondria: carbodiimide-catalyzed acylation of a functional lysine residue.
Upon modification of the reconstituted aspartate/glutamate carrier by various amino acid-reactive chemicals a functional lysine residue at the exofacial binding site was identified. The inactivation of transport function by the lysine-specific reagents pyridoxal phosphate (PLP, IC50 400 microM) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS, IC50 300 microM) could specifically be suppressed by the substrates aspartate and glutamate; a 50% substrate protection was observed at half-saturation of the external binding site. The same held true for 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, IC50 500 microM) and diethyl pyrocarbonate (DEPC, IC50 20 microM), two reagents known to modify carboxylic or histidinyl side-chains, respectively. EDC, however, turned out to catalyze an acylation of the active site lysine by activating carboxyls that had to be present in the incubation medium. This special mechanism, which was proven by protein labelling using EDC/[14C]succinate, necessitates a lysine side-chain of high reactivity and low pK, since the modification had to occur at pH less than or equal to 6.5, i.e. not too far from the pK of the carboxyl to be activated. All reagents applied, additionally including 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS, IC50 10 microM), were effective at this pH. Competition experiments revealed interaction of EDC, PLP, SITS and probably DIDS at the same active site lysine. For DEPC a lysine modification could not be ruled out. Yet, a model comprising a histidine juxtaposed to the lysine seems to be appropriate.
Dierks
T
T
Institut für Biotechnologie, Forschungszentrum Jülich, Germany.
Stappen
R
R
Salentin
A
A
Krämer
R
R
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000378:metabolism; D000215:Acylation; D026922:Amino Acid Transport Systems, Acidic; D000818:Animals; D017920:Antiporters; D001224:Aspartic Acid / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002384:Catalysis; D002417:Cattle; D004047:Diethyl Pyrocarbonate / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism*; D011510:Proteolipids; D011732:Pyridoxal Phosphate / Q000378:metabolism
D026922:Amino Acid Transport Systems, Acidic; D017920:Antiporters; D002352:Carrier Proteins; D005971:Glutamates; D011510:Proteolipids; C051046:aspartate-glutamate carrier; C020485:proteoliposomes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D001224:Aspartic Acid; D018698:Glutamic Acid; D011732:Pyridoxal Phosphate; D008239:Lysine; D004047:Diethyl Pyrocarbonate; D005022:Ethyldimethylaminopropyl Carbodiimide