Site-directed alteration of three 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 Km for histidine of 50 mM, while the relaxed (R) state has a Km 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 Km and in kcat 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 Km 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 kcat, but raised the Km 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.
Pishko
E J
EJ
Biochemical Institute, University of Texas, Austin 78712.
Robertus
J D
JD
D000409:Alanine; D000494:Allosteric Regulation; D001216:Asparagine; D001665:Binding Sites; D006639:Histidine / Q000378:metabolism*; D006640:Histidine Decarboxylase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D011773:Pyruvates / Q000378:metabolism*; D019289:Pyruvic Acid; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine; D014633:Valine
D011773:Pyruvates; D011994:Recombinant Proteins; D007532:Isoleucine; D014443:Tyrosine; D010649:Phenylalanine; D006639:Histidine; D001216:Asparagine; D019289:Pyruvic Acid; D006640:Histidine Decarboxylase; D014633:Valine; D000409:Alanine