| Abstract: | The fragmentary form of alanine racemase from Bacillus stearothermophilus is composed of two sets of two different polypeptides corresponding to the two domains of the subunit of wild-type enzyme. It was denatured with 6 M guanidium hydrochloride to be separated into pieces, and renatured by dilution with about 50% recovery of the activity. The two kinds of polypeptides (i.e., large and a small fragments) were isolated by gel filtration in the presence of 4 M guanidium hydrochloride. The CD spectra obtained by summation of the spectra of the refolded fragments were closely similar to that of the native fragmentary enzyme. The lysine residue to which PLP is bound in the wild-type enzyme occurs in the large peptide of the fragmentary enzyme containing the amino terminus of the wild-type enzyme. The visible spectrum of the large peptide refolded, however, indicated that PLP was not bound to it. The large peptide alone had no significant activity, but it was activated by incubation with the small peptide. Accordingly, co-existene of both peptide fragments is necessary for folding of a complex of the two kinds of peptide to form an active structure. |
