Catalytic Properties of Enzymes from Erwinia carotovora Involved in Transamination of Phenylpyruvate

摘要

K-m for L-phenylalanine, L-glutamic acid, L-aspartic acid, and the corresponding keto acids were calculated, as well as V-max was measured for the following pairs of substrates: L-phenylalanine-2-ketoglutarate, L-phenylalanine-oxaloacetate, L-glutamic acid-phenylpyruvate, and L-aspartic acid-phenylpyruvate for aminotransferases PAT1, PAT2, and PAT3 from Erwinia carotovora catalyzing transamination of phenylpyruvate. The ping-pong bi-bi mechanism was shown for the studied aminotransferases. The substrate inhibition (K-s) of PAT3 with 2-ketoglutarate and oxaloacetate was 10.23 +/- 3.20 and 3.73 +/- 1.99 mM, respectively. It was shown that L-beta-(N-benzylamino) alanine was a competitive inhibitor with respect to L-phenylalanine for PAT1 (K-i = 0.32 +/- 0.07 mM, K-m = 0.45 +/- 0.1 mM, V-max = 11. 6 +/- 0.4 U/mg) at 25 mM concentration of 2-ketoglutarate in the reaction medium. L-beta-(N-methylamino) alanine is a noncompetitive inhibitor with respect to L-phenylalanine for PAT3 (K-I = 138.4 +/- 95.4 mM, Km = 13.7 +/- 3.9 mM, V-max = 18.6 +/- 4.1 U/mg) at 2 mM concentration of 2 - ketoglutarate in the reaction medium. L-stereo isomers of nonprotein analogues of aromatic amino acids were studied as substrates for PAT1, PAT2, and PAT3. L-beta-(2-Br-phenyl) alanine, L-beta-(4-Br-phenyl) alanine, L-beta-(2-F-phenyl) alanine, and L-(2-F) tryptophan were good substrates for all three aminotransferases; L-alpha-methyl-beta-(2-Br-phenyl) alanine and L-O-benzyltyrosine were substrates only for PAT3; L-beta-(4-F-phenyl) alanine was a substrate for PAT1 and PAT3. Thus, these analogues of aromatic amino acids can be stereoselectively synthesized using the studied aminotransferases in the presence of the corresponding keto acids.