Identification of the Subunit and Important Target Peptides of Pig Heart NAD-Dependent Isocitrate Dehydrogenase Modified by the Affinity Label Adenosine 5‘-O-[S-(4-Bromo-2,3-dioxobutyl)thiophosphate]

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Abstract
Pig heart NAD-dependent isocitrate dehydrogenase is inactivated by adenosine 5‘-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate] (AMPS-BDB) with incorporation of 1.78 mol of reagent/mol of average subunit. Complete protection against the inactivation is provided by 20 mM isocitrate + 1 mM Mn2+, and the incorporation is decreased to about 1.3 mol of reagent/mol of average subunit. The addition of NAD, NADH, or Mn2+ alone has little effect on the functional changes produced by AMPS-BDB, while ADP gives only partial protection against the inactivation. The ability of ADP to decrease the Km for isocitrate is not affected by the AMPS-BDB modification of the enzyme. These results indicate that the isocitrate substrate site is the target of AMPS-BDB. The enzyme has three types of subunits with a tetramer having the composition α2βγ. Here, [2-3H]AMPS-BDB-modified subunits are separated by HPLC on a C4 reverse-phase column, after the treatment of the modified enzyme with 4 M urea. The predominant radioactivity is distributed in α and γ subunits. However, evidence based on recombination of subunits from modified and unmodified enzymes indicates that only labeling of the α subunit is responsible for inactivation by AMPS-BDB. Subsequently, the separated modified subunits were chemically cleaved by CNBr and then purified by HPLC using a C18 column. The labeled peptides were further digested by pepsin, purified by HPLC, and sequenced. These results indicate that R88 and R98 from the α subunit are the major targets of AMPS-BDB which cause inactivation and that these are at or near the isocitrate site of the enzyme.