Properties of aspartate racemase, a pyridoxal 5'-phosphate-independent amino acid racemase.

Abstract

Aspartate racemase from Streptococcus thermophilus contains no pyridoxal 5"phosphate or other cofactors such as FAD, NAD+, and metal ions.It was affected by neither carbonyl reagents such as hydroxylamine nor sodium borohydride but was strongly inhibited by iodoacetamide and other thiol reagents.Aspartate, cysteate, and cysteine sulfinate were the only substrates.The K , values for L-and D-aspartate were 35 and 8.7 mM, respectively.The enzyme catalyzed the exchange of a-hydrogen of the substrate with the solvent hydrogen.Racemization of L-aspartate in 2Hz0 showed an overshooting in the optical rotation of aspartate before the substrate was fully racemized.This shows that the removal of a-hydrogen of the substrate is at least partially rate-determining.When L-or D-aspartate was incubated with aspartate racemase in tritiated water, tritium was incorporated preferentially into the product enantiomer.The results strongly suggest that aspartate racemase contains two hydrogen acceptors.Racemization is superficially a simple reaction.For example, it is accomplished by the removal of a-hydrogen bound to a chiral carbon of the substrate and subsequent nonspecific return of a hydrogen to the carbon.However, amino acids are racemized only slowly under ordinary conditions: half-lives of aspartic acid and alanine in the racemization at 25 "C are 3,500 and 12,000 years, respectively (Bada, 1985).This is because of the high dissociation energy of the C"-H bond.Amino acid racemases and epimerases catalyze the racemization and epimerization of amino acids, respectively, and most of them require PLP' as a coenzyme.The racemization proceeds through the formation of aldimine Schiff base between the substrate amino acid and PLP (Snell and Di Mari, 1970).However, several other amino acid racemases require no coenzymes.Proline racemase (EC 5.1.1.4)catalyzes the proline racemization effectively without PLP by action of a pair of cysteinyl residues at the active site of the enzyme (Cardinale and Abeles, 1968;Rudnick and Abeles, 1975).Knowles and his co-workers studied the energetics and mechanism of the enzyme reaction (Albery and Knowles, 1986, 1987a, 1987b; Fisher et al., 1986a Fisher et al., , 1986b Fisher et al., , 1986c;; Belasco et al.,