Stereospecificity of reactions catalyzed by bacterial D-amino acid transaminase.
The spectral shift from 420 to 338 nm when pure bacterial D-amino acid transaminase binds D-amino acid substrates is also exhibited in part by high concentrations of L-amino acids (L-alanine and L-glutamate) but not by simple dicarboxylic acids or monoamines. Slow processing of L-alanine to D-alanine was observed both by coupled enzymatic assays using D-amino acid oxidase and by high pressure liquid chromatography analysis employing an optically active chromophore (Marfey's reagent). When the acceptor for L-alanine was alpha-ketoglutarate, D-glutamate was also formed. This minor activity of the transaminase involved both homologous (L-alanine and D-alanine) and heterologous (L-alanine and D-glutamate) substrate pairs and was a function of the nature of the keto acid acceptor. In the presence of alpha-ketoisovalerate, DL-alanine was almost completely processed to D-valine; within the limits of the assay no L-valine was detected. With alpha-ketoisocaproate, 90% of the DL-alanine was converted to D-leucine. In the mechanism of this transaminase reaction, there may be more stereoselective constraints for the protonation of the quinonoid intermediate during the second half-reaction of the transamination reaction, i.e. the donation of the amino group from the pyridoxamine 5'-phosphate coenzyme to a second keto acid acceptor, than during removal of the alpha proton in the initial steps of the reaction pathway. Thus, with this D-amino acid transaminase, the discrete steps of transamination ensure fidelity of the stereospecificity of reaction pathway.
Martínez del Pozo
A
A
Rockefeller University, New York, New York 10021.
Merola
M
M
Ueno
H
H
Manning
J M
JM
Tanizawa
K
K
Nishimura
K
K
Soda
K
K
Ringe
D
D
D001407:Bacillus / Q000201:enzymology*; D051263:D-Alanine Transaminase; D007700:Kinetics; D013237:Stereoisomerism; D013379:Substrate Specificity; D000637:Transaminases / Q000378:metabolism*
D000637:Transaminases; D051263:D-Alanine Transaminase