Abstract：
: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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Pishko EJ,Robertus JDdoi
10.1021/bi00069a032subject
Has Abstractpub_date
1993-05-11 00:00:00pages
4943-8issue
18eissn
0006-2960issn
1520-4995journal_volume
32pub_type
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