EVIDENCE FOR CONFORMATIONAL-CHANGES IN ESCHERICHIA-COLI PORPHOBILINOGEN DEAMINASE DURING STEPWISE PYRROLE CHAIN ELONGATION MONITORED BY INCREASED REACTIVITY OF CYSTEINE-134 TO ALKYLATION BY N-ETHYLMALEIMIDE

Porphobilinogen deaminase from Escherichia coli becomes progressively more susceptible to inactivation by the thiophilic reagent N-ethylmale imide (NEM) as the catalytic cycle proceeds through the enzyme-interme diate complexes ES, ES(2), ES(3), and ES(4). Site-directed mutagenesis of potentially reactive cysteines has been used to identify cysteine- 134 as the key residue that becomes modified by the reagent and leads to inactivation. Since cysteine-134 is buried at the interface between domains 2 and 3 of the E. coli deaminase molecule, the observations s uggest that a stepwise conformational change occurs between these doma ins during each stage of tetrapyrrole assembly. Interestingly, mutatio n of the invariant active-site cysteine-242 to serine leads to an enzy me with up to a third of the catalytic activity found in the wild-type enzyme. Electrospray mass spectrometry indicates that serine can subs titute for cysteine as the dipyrromethane cofactor attachment site.