SITE-DIRECTED MUTAGENESIS OF INTERSUBUNIT BOUNDARY RESIDUES IN HISTIDINE-DECARBOXYLASE, A PH-DEPENDENT ALLOSTERIC ENZYME

Histidine decarboxylase (HDC) from Lactobacillus 30a forms a trimer ar ound a central cavity or well. Three active sites are formed around th e well at the interface of each of two adjacent molecules. HDC exhibit s cooperative kinetics at pH 7.6 and can be described in terms of a tw o-state, T and R, model. At pH 4.8, protons stabilize HDC in the R for m. Asp 198 and Asp 53, from a neighboring molecule, are the core of th e pH-sensitive mechanism controlling the shift in quaternary state. Ei ght site-directed mutations have been made to analyze the region. Seve ral mutants, including the conversion of Asp 53 to Asn, cause HDC to e xhibit sigmoidal kinetics even at pH 4.8. Others lock the enzyme into the T state. Kinetic analysis suggests that k(cat) values for T and R states are similar. The K-m for the T state, near 8 mM, exceeds that f or the R state by 40-fold and shows HDC is primarily regulated by alte ring its affinity for substrate.