Activities | Families | Sequences | Fold types | References | Help
B6db references: 93075786

type Journal Article
authors Yoshimura, T.; Bhatia, M. B.; Manning, J. M.; Ringe, D.; Soda, K.
title Partial reactions of bacterial D-amino acid transaminase with asparagine substituted for the lysine that binds coenzyme pyridoxal 5'- phosphate
journal Biochemistry
ui 93075786
year (1992)
volume 31
number 47
pages 11748-54.
keywords Alanine/pharmacology
abstract In bacterial D-amino acid transaminase (EC replacement of Lys- 145, which is covalently linked to the coenzyme pyridoxal 5'-phosphate in the wild-type enzyme, by an Asn residue gave a mutant enzyme (K145N) that slowly performed each half-reaction, as determined by spectral measurements. With the wild-type enzyme, the kinetics of these events were so rapid that pre-steady-state conditions were needed for their determination. The internal aldimine between coenzyme and Lys-145 was rapidly reduced with NaCNBH3 in the wild-type enzyme, whereas in the mutant enzyme the coenzyme, which is not covalently linked to the protein, was more resistant to reduction; the reduced forms of both wild-type and mutant enzymes were inactive. With large amounts of the K145N mutant enzyme and either amino acid or keto acid substrate alone, the formation of some reaction intermediates, i.e., the external aldimine with D-alanine and the ketimine with alpha-ketoglutarate, can be measured by conventional spectroscopy. Suicide substrates also induced slow spectral shifts of the E-PLP form of the enzyme. For the K145N enzyme, exogenous amines affected only the rate of the transaldimination but not the removal of the alpha-proton of the substrate. These results suggest that in the mutant enzyme some amino acid side chain other than Lys-145 performs this function. In order to identify this site, the K145N mutant enzyme was completely inactivated by the radiolabeled suicide substrate D-serine. Peptide mapping of tryptic digests showed that Lys-267 was the modified site.(ABSTRACT TRUNCATED AT 250 WORDS)
last changed 2002/11/04 17:41

B6db references