SITE-DIRECTED ALTERATION OF 3 ACTIVE-SITE RESIDUES OF A PYRUVOYL-DEPENDENT HISTIDINE-DECARBOXYLASE

The active site of histidine decarboxylase (HDC) from Lactobacillus 30a contains a pyruvoyl cofactor sitting at the interface of two molecules in a trimer. Although exhibiting hyperbolic kinetics at pH 4.8, near its optimum, HDC is cooperative at pH 7.6, indicating that the units of the trimer communicate. A Hill plot analysis shows that HDC, at pH 7.6, can be described by a two-state model. The tense (T) state has an apparent K(m) for histidine of 50 mM, while the relaxed (R) state has a K(m) of 5 mM. To explore the catalytic mechanism, three of the cross-boundary active-site residues were altered by site-directed mutagenesis and their effects observed. Ile-59 is known to act as lid on the substrate binding pocket; it was converted to Ala (I59A) and to Val (I59V). The former was inactive, attesting to the importance of this residue in the mechanism. The I59V mutant showed a decrease in K(m) and in k(cat) at pHs 4.8 and 7.6. Ile-59 appears to help orient substrate properly for catalysis; decreasing its size expands the binding site. This may allow the substrate to bind more readily, but in a number of conformations which are not optimal for catalysis. Conversion of Tyr-62 to Phe (Y62F) had no effect on catalysis but raised the K(m) 7-fold at pH 4.8. Asp-63 appears to form an ion pair to the substrate imidazolium. Conversion to the neutral amide (D63N) had no effect on the k(cat), but raised the K(m) 240-fold at pH 4.8. This is consistent with the notion that the ion pair is up to 3 kcal/mol stronger than a simple hydrogen bond with the substrate. The mutant had no detectable activity at pH 7.6.