|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1993 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1992 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Two distinct types of amino acid racemases are known : one requires pyridoxal phosphate as a coenzyme and is represented by alanine racemase, the other requires neither cofactors nor coenzymes and is represented by glutamate racemase. It is interesting and important to compare catalytic mechanisms of these two types of enzymes, both of which catalyze racemization of alph-amino acids. We aimed to study the structures and functions of both types of enzymes, thereby to clarify mechanisms of the amino acid racemization. As to pyridoxal phosphate dependent amino acid racemases, we have chosen thermostable alanine racemase from Bacillus stearothermophilus. We have constructed a mutant enzyme of it in which Lys39 binding the coenzyme pyridoxal phosphate is replaced by alanlne, purified it, and characterized its enzymological properties. In particular, we have studied spectrophotometric properties of the coenzyme complexes and reaction with beta-chloroalanine. Pyridoxal-dependent amino acid racemase with low substrate specificity from Pseudomonas putida has been studied as well. We have cloned its structural gene, purified the enzyme from the clone cells, characterized the enzyme, determined the stereochemistry of hydrogen transfer in the abortive transamination, and studied the reaction mechanism. On the other hand, as to pyridoxal phosphate-independent racemases, glutamate racemase and aspartate racemase, we studied the role of cysteine residues of both enzymes by site-directed mutagenesis and chemical modification. Glutamate racemase loses the ability to transfer alph-proton, when either Cys73 or Cys184 is replaced by Ala or modified. On the other hand, apsartate racemase is completely inactivated by modification or replacement of only one Cys73 per dimeric enzyme.
Therefore, we have proposed that asparate racemase probably has a composit active site.