Abstract
Limited proteolysis studies on alanine racemase suggested that the enzyme subunit is composed of two domains (Galakatos, N. G., and Walsh, C. T. (1987) Biochemistry 26, 8475-8480). We have constructed a mutant gene that tandemly encodes the two polypeptides of the Bacillus stearothermophilus enzyme subunit cleaved at the position corresponding to the predicted hinge region. The mutant gene product purified was shown to be composed of two sets of the two polypeptide fragments and was immunologically identical to the wild-type enzyme. The mutant enzyme, i.e. the fragmentary alanine racemase, was active in both directions of the racemization of alanine. The maximum velocity (Vmax) was about half that of the wild-type enzyme, and the Km value was about double. Absorption and circular dichroism spectra of the fragmentary enzyme were similar to those of the wild-type enzyme. An attempt was made to separately express in Escherichia coli a single polypeptide corresponding to each domain, but no protein reactive with the antibody against the wild-type alanine racemase was produced. Therefore, it is suggested that the two polypeptide fragments can fold into an active structure only when they are co-translated and that they correspond to structural folding units in the parental polypeptide chain.
Highlights
From the Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 61I and the flaboratom ofFood Chemistm. the Institute of Scientific and Industrial Research,Osaka University, Mihogaoka 8-1, Ibaraki
Limited proteolysis studies on alanine racemase sug- Recently, we have cloned the gene for thermostable alanine gested that the enzyme subunit is composed of two racemase from the Bacillus stearothermophilus, purified the domains
We have constructed a mutant gene that tandemly encodes the two polypeptides of the Bacillus stearothermophilus enzyme subunit cleaved at the position corresponding to the predicted hinge region
Summary
We have constructed a mutant gene that tandemly encodes the two polypeptides of the Bacillus stearothermophilus enzyme subunit cleaved at the position corresponding to the predicted hinge region. Galakatos and Walsh (1987)reported that both DadB and Alr isozymes of alanine racemase of S. typhimurium suffered from limited proteolysis at two homologous positions by trypsin, chymotrypsin, or subtilisin generating two nonoverlapping polypeptides with M , 28,000 and 11,000 and a tetrapeptide, which wasregarded as ahinge region. It is suggested that the two polypeptide only 35 and 31% sequence homology with DadB and Alr fragments can fold into an active structure only when enzymes, respectively, local sequences including the active they are co-translated and that they correspond to site and hinge regions are well conserved. The gene was expressed as an active alanine racemase composed of two dissimilar polypeptides
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