Effect of enzyme inhibitors on protein quaternary structure determined by on-line size exclusion chromatography-microelectrospray ionization mass spectrometry

Aldehyde dehydrogenases (ALDH) are a family of enzymes primarily involved in the oxidation of various aldehydes. Most ALDH enzymes derived from mammalian sources have been shown to exist as homotetramers, consisting of four identical subunits of approximately 54 kDa. The presence of the homotetramer appears to be necessary for enzyme activity. In this study, recombinant rat liver mitochondrial ALDH (rmALDH) was inhibited in vitro with four different inhibitors, namely, disulfiram (MW, 296.5), prunetin (MW, 284.3), benomyl (MW, 290.3), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (MW, 351.8). Subsequently, inhibited rmALDH was analyzed by a novel approach of on-line size exclusion chromatography-microelectrospray ionization-mass spectrometry (SEC-mu ESI-MS) to examine the noncovalent quaternary structural stability of the inhibited enzyme. Analysis of native rmALDH by SEC-mu ESI-MS revealed predominantly the homotetramer (M-r = similar to 217,457 Da, +/-0.01%) with some in-source, skimmer-induced dissociation to afford monomer (M-r = similar to 54,360 Da, +/-0.01%). Both disulfiram and prunetin inhibited rmALDH by >70% and >90%, respectively, but did not disrupt the quaternary structure of rmALDH. Furthermore, there was no detectable change within experimental error (+/-0.01%) of the disulfiram or the prunetin homotetramers (M-r = similar to 217,448 Da and M-r = similar to 217,446 Da). This may possibly indicate that inhibition occurred via formation of intramolecular disulfide bond at the enzyme active site, or weak affinity noncovalent binding. In contrast, benomyl-inhibited rmALDH homotetramer (>90% inhibition) exhibited a M-r = similar to 217,650 Da (+/-0.01%) corresponding to two butylcarbamoyl adducts on two of the four enzyme subunits. The skimmer-induced monomer afforded a mixture of unmodified rmALDH (M-r = similar to 54,365 Da, +/-0.01%) and butylcarbamoylated enzyme (M-r = similar to 54,459 Da, +/-0.01%). Finally, TPCK (>90% inhibition) modified all four subunits of rmALDH to give M-r = similar to 218,646 Da (+/-0.01%). In all four cases while significant enzyme inhibition occurred, no destabilization of the quaternary complex was detected. (C) 2001 American Society for Mass Spectrometry.