Imino acid dehydrogenases

In an original application, Yasuda et al have used both l-AAO and d-AAO, and L-lysine oxidase to oxidize o ,Ci -diamino acids. The reactions produce the expected a-keto w-amino acid products, but these then spontaneously cyclize to form cyclic a-imino acids. These compounds are then substrates for the authors recently discovered A methyl amino acid dehydrogenase (NMAADH) from Pseudomonas putida, producing the pure L-cyclic amino acid (Scheme 5). 

The availability of stable isotope-labeled PA makes an accurate quantitative determination of this imino acid possible. A short high-performance liquid chromatography (HPLC) run prior to the mass spectrometer inlet will result in a discrete peak of PA. For the definitive diagnosis of AASA dehydrogenase deficiency, a simultaneous determination of AASA would be preferred. The absence of a commercially available labeled standard leaves this analysis in the experimental stage. 

The only preparative-scale reactions of synthetic value in this category are those catalyzed by the amino acid dehydrogenases. These enzymes catalyze the reductions of a-imino acids to a-amino acids. This can be done on a very large scale, as demonstrated by the LeuDH-catalyzed reduction of (120) to f-butyl leucine (121) shown in Scheme 59. Another enzyme of this group with preparative promise is... 

Glutamate dehydrogenase plays a major role in amino acid metabolism. It is a zinc protein, requires NAD+ or NADP+ as coenzyme, and is present in high concentrations in mitochondria of liver, heart, muscle, and kidney. It catalyzes the (reversible) oxidative deamination of L-glutamate to a-ketoglutarate and NH3. The initial step probably involves formation of a-iminoglutarate by dehydrogenation. This step is followed by hydrolysis of the imino acid to a keto acid and NH3 ... 

The reaction depends upon all of the components shown in equation (I). It has been proposed that an imino acid is an intermediate, but no evidence for the existence of such a compound has been reported. If a nonenzymatic reaction between ammonia and a-ketoglutarate were to form the true substrate, the apparent requirements for enzyme saturation should be equivalent for both compounds and the apparent requirement for each should depend on the concentration of the other, because the hypothetical imino acid would be formed in a second-order reaction of ammonia and the keto acid. Instead, a Km of 1.2 X 10 was found for a-ketoglutarate and 5.7 X 10 for NH4. Other Km values for this enzyme are 2 X 10 for glutamate, 2.5 X 10 for DPN, and 1.8 X 10 for DPNH. These values are influenced by ions in the medium and pH, and are not fixed properties of the enzyme. As with other enzymes that use both DPN and TPN, glutamic dehydrogenase also acts with desamino DPN and acetylpyridine DPN. 

Thus, the primary oxidative step is a dehydrogenation (and thus the term dehydrogenase is preferable) to form an imino acid which in turn is non-enzymatically hydrolyzed to form the keto acid and ammonia or a derivative thereof. In the formulation of reactions 2 and 3, Ri is intended to represent either a hydrogen atom or a substituent, such as an alkyl group. If Ri is a hydrogen atom, ammonia is formed if Ri is an alkyl group, an alkyl amine is formed. 

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