BINDING TO PHOSPHOLIPID-VESICLES IMPAIRS SUBSTRATE-MEDIATED CONFORMATIONAL-CHANGES OF THE PRECURSOR TO MITOCHONDRIAL ASPARTATE-AMINOTRANSFERASE

Specific labeling of both the mature (mAspAT) and precursor (pmAspAT) forms of rat liver mitochondrial aspartate aminotransferase with three different spectroscopic probes (monobromotrimethylammoniobimane, N-(i odoacetylaminoethyl)-5-naphthalene-1-sulfonic acid, and N-(1-pyrenyl)m aleimide) was used to assess the possible conformational consequences of the interaction of a mitochondrial precursor protein with lipid mem branes by means of fluorescence spectroscopy. The three probes react w ith the same cysteine residue causing a partial loss of catalytic acti vity whose extent depends on the nature of the probe introduced. The f luorescence intensity of the attached probes decreases upon addition o f substrates or substrate analogues, indicating that the modified enzy mes can undergo the open-closed conformational transitions that accomp any catalysis. Both unmodified and labeled precursor proteins bind to negatively charged phospholipid vesicles, whereas the mature enzyme is unable to bind. Binding to liposomes does not affect the fluorescent properties of the attached probes. However, addition of the pseudosubs trate alpha-methylaspartate to liposome-bound precursor fails to induc e the characteristic conformational changes observed with the protein free in solution. Furthermore, upon binding to liposomes the precursor protein loses enzymatic activity, and the reactive cysteine residue b ecomes inaccessible to reaction with thiol reagents. In contrast, the presence of liposomes has no effect on the activity, cysteine reactivi ty, or syncatalytic conformational transitions of the mature enzyme. I t appears that interaction of pmAspAT with negatively charged phosphol ipids prevents the protein from undergoing the conformational transiti ons required for catalysis, ''freezing'' the enzyme in a sterically hi ndered but open-like conformation.