Kinetic mechanism of Bacillus subtilis L-alanine dehydrogenase

Publisher Website
Google Scholar
Abstract
L-Alanine dehydrogenase from B. subtilis has a predominantly ordered kinetic mechanism in which NAD adds before L-alanine, and ammonia, pyruvate and NADH are released in that order. When pyruvate is varied at pH 9.35, levels of ammonia above 50 mM cause uncompetitive substrate inhibition and cause the slope replot to go through the origin. This pattern suggests that iminopyruvate (2% of pyruvate at this pH with 150 mM ammonia) can combine with the enzyme (E)-NADH complex much more tightly than pyruvate does but reacts much more slowly because uptake of the required proton from solution is hindered. Isomerization of the initially formed E-NAD complex to a form which can productively bind L-alanine is the slowest step in the forward direction at pH 7.9, and substrate inhibition by L-alanine largely results from combination of the zwitterion in a nonproductive fashion with this initial E-NAD complex, with the result that the isomerization is prevented. All bimolecular rate constants approach diffusion-limited values of optimal states of protonation of enzyme and substrates except that for ammonia, suggesting that ammonia does not form a complex with E-NADH-pyruvate but reacts directly with it to give a bound carbinolamine.