FEE,
HEGEMAN,
A N D KENYON
Mandelate Racemase from Pseudomonas putida. Subunit Composition and Absolute Divalent Metal Ion Requirementt Judy A. Fee, George D. Hegeman,$ and George L. Kenyon*!§
ABSTRACT : Polyacrylamide gel electrophoresis of mandelate racemase (EC 5.1.2.2) in the presence of sodium dodecyl sulfate gives a single protein band corresponding to a molecular weight of ca. 69,500. Four protein bands, indicative of four subunits, result from cross-linking of the enzyme with dimethyl suberimidate followed by sodium dodecyl sulfate gel electrophoresis. These and earlier results [Hegeman, G. D., Rosenberg, E. Y . , and Kenyon, G. L. (1970), Biochemistry 9, 40291 suggest that the enzyme is composed of four identical subunits and has a molecular weight of ca. 278,000. The enzyme is completely inactivated when incubated with excess EDTA. Moreover, exhaustive efforts to remove contaminating di-
P
revious structural studies on mandelate racemase (Hegeman et al., 1970; Hegeman, 1970) had led to the postulate that the enzyme was composed of identical subunits, and a molecular weight of ca. 200,000 was estimated for the oligomer from gel filtration experiments. In this paper we report our findings using the more reliable technique of gel electrophoresis in the presence of sodium dodecyl sulfate (Weber and Osborn, 1969), which shows a single staining band for the protein. Knowledge about subunit composition and molecular weight was essential to determining the stoichiometry of affinity labeling of the enzyme by DL-a-phenylglycidate, discussed in an accompanying paper (Fee et al., 1974). We have also reinvestigated the divalent metal ion requirement for optimal enzyme activity and report explicit instructions for removal of contaminating divalent metal ions from the enzyme and assay mixture. Weil-Malherbe (1966) has previously reported that divalent metal ions, e.g., Mg2+, stimulate binding of substrate. We find here that with great care in divalent metal ion removal completely inactive enzyme can be produced. Experimental Section Materials and Methods. General. Both mandelate racemase and L-mandelate dehydrogenase were prepared and assayed as previously described (Hegeman et al., 1970; Hegeman, 1970). Protein was measured by the method of Lowry et al. (1 951). Samples were compared to standard solutions of i.From the Departments of Chemistry, and Bacteriology and Immunology, University of California, Berkeley, California 94720, the Departments of Pharmaceutical Chemistry and Biochemistry and Biophysics, University of California, San Francisco, California 94143, and the Department of Microbiology, Indiana University, Bloomington, Indiana 47401. Receiced January 31, 1974. This work was supported by U. S. Public Health Service Grants AM-17323 from the National Institute of Arthritis, Digestive Diseases and Metabolism (G.L. I
