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 i n the oxidation of various aldehydes. Most ALDH enzymes derived from mammal ian sources have been shown to exist as homotetramers, consisting of four i dentical subunits of approximately 54 kDa. The presence of the homotetramer appears to be necessary for enzyme activity. In this study, recombinant ra t liver mitochondrial ALDH (rmALDH) was inhibited in vitro with four differ ent inhibitors, namely, disulfiram (MW, 296.5), prunetin (MW, 284.3), benom yl (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 sp ectrometry (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 monome r (M-r = similar to 54,360 Da, +/-0.01%). Both disulfiram and prunetin inhi bited rmALDH by >70% and >90%, respectively, but did not disrupt the quater nary structure of rmALDH. Furthermore, there was no detectable change withi n experimental error (+/-0.01%) of the disulfiram or the prunetin homotetra mers (M-r = similar to 217,448 Da and M-r = similar to 217,446 Da). This ma y possibly indicate that inhibition occurred via formation of intramolecula r disulfide bond at the enzyme active site, or weak affinity noncovalent bi nding. In contrast, benomyl-inhibited rmALDH homotetramer (>90% inhibition) exhibited a M-r = similar to 217,650 Da (+/-0.01%) corresponding to two bu tylcarbamoyl adducts on two of the four enzyme subunits. The skimmer-induce d 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 rmAL DH to give M-r = similar to 218,646 Da (+/-0.01%). In all four cases while significant enzyme inhibition occurred, no destabilization of the quaternar y complex was detected. (C) 2001 American Society for Mass Spectrometry.