Abstract
L-alanine dehydrogenase (AlaDH, EC 1.4.1.1) from Bacillus subtilis catalyzes the biotransformation of β-fluoropyruvic acid into β-fluoro-L-alanine in presence of cofactor NADH and ammonia. This unnatural β-fluorinated amino acid shows antibacterial properties due to inactivation of alanine racemase - enzyme involved in peptidoglycan biosynthesis in bacterial cell wall. We report the studies on mechanism of this reaction using solvent, SIE, and kinetic, KIE, isotope methods. The kinetic of reaction was studied spectrophotometrically by measuring the decreasing absorbance of the reduced form of NADH at 340 nm. For kinetic studies the deuterium labeled NADH was obtained by reduction of NAD + catalyzed by formate dehydrogenase (FDH, EC 1.2.1.2). SIE’s and KIE’s were determined using non-competitive spectroscopic method.
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