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Inhibition and Kinetic Isotope Effect Studies to Further Characterize the Mechanism of Maleamate Amidohydrolase
Author(s) -
Malani Karan,
Snider Mark
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.572.13
Subject(s) - kinetic isotope effect , chemistry , amidohydrolase , stereochemistry , reaction mechanism , catalysis , enzyme , medicinal chemistry , organic chemistry , deuterium , physics , quantum mechanics
Maleamate amidohydrolase (NicF) is involved in the degradation of nicotinic acid, catalyzing the deamination of maleamate to form maleate. To provide further insight concerning the mechanism of this enzymatic reaction, kinetic and UV spectroscopy assays were conducted to characterize the binding of (Z)‐4‐oxobut‐2‐enoic acid by NicF. The results indicate that this molecule behaves as a reversible competitive inhibitor with a K i of 1.3 (± 0.13) x 10 ‐8 M. Overall, (Z)‐4‐oxobut‐2‐enoic acid was shown to be bound 10 x more tightly than the previously studied inhibitor maleic anhydride, and 400,000 times more tightly than the product, maleate. The observed tight binding of this aldehyde inhibitor is consistent with the proposed addition‐elimination mechanism for catalysis. Additionally, D 2 O kinetic isotope effects were measured to determine the number of protons essential for the rate limiting step. The D k cat was observed to be insensitive to increasing mole fraction of D 2 O, whereas D k cat / K M was inverse, increasing exponentially with increasing D 2 O. Thus, substrate binding appears to be accompanied by at least 3 proton transfers leading up to the first irreversible step.