Site-directed alteration of serine 82 causes nonproductive chain cleavage in prohistidine decarboxylase.

Prohistidine decarboxylase from Lactobacillus 30a normally autoactivates by cleavage of the Ser-81-Ser-82 peptide bond, converting Ser-82 to a pyruvoyl moiety which serves as the enzymatic cofactor. We have used site-directed methods to make two conservative mutations, converting Ser-82 to cysteine (S82C) and threonine (S82T). Both mutant proteins autoactivate, although dramatically (20- to 80-fold), more slowly than wild type. Roughly 55% of the mutant protein in each case undergoes a nonproductive chain cleavage which does not result in cofactor production. This finding suggests that an important feature in the enzyme's evolution has been development of an activation scheme which minimizes nonproductive side reactions. Catalytic constants are also affected by the mutations, particularly kcat which drops 8-fold in S82C and 450-fold in S82T. In addition, the S82T protein activates to produce a novel alpha-ketobutyroyl cofactor.