Inhibition of glyceraldehyde-3-phosphate dehydrogenase by pentalenolactone. 2. Identification of the site of alkylation by tetrahydropentalenolactone.

Incubation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the antibiotic pentalenolactone (3) results in time-dependent, irreversible inhibition of GAPDH by modification of a single Cys residue in each subunit of the homotetrameric enzyme. Reduction of pentalenolactone with tritium gas gave [2,3,7,8-3H4]tetrahydropentalenolactone (7), which also exhibited time-dependent, irreversible inactivation of GAPDH. The site of covalent attachment of 7 was determined. Tryptic digestion of inactivated GAPDH and purification of the resultant products by reverse-phase HPLC gave a single labeled peptide. Amino acid sequence analysis of the radioactive peptide gave Ile-Val-Ser-Asn-Ala-Ser-X-Thr-Thr-Asn-(...). This sequence is identical to the highly conserved region from Ile-143 to Asn-152 in pig muscle GAPDH, except for the active site Cys-149 to which the tetrahydropentalenolactone was covalently bound. Molecular modeling was used to compare both pentalenolactone (3) and heptelidic acid (4), a mechanistically related inactivator of GAPDH, with the normal substrate, glyceraldehyde 3-phosphate (1). Finally, pentalenolactone was shown by reaction with model thiols to undergo epoxide ring opening exclusively by nucleophilic attack at the primary carbon, C-10.