Physical and chemical evidence for the trimeric subunit structure of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli K-12.

Multiple lines of evidence are presented which support the conclusion that 2-keto-4-hydroxyglutarate aldolase, obtained in homogeneous form (over 2,000-fold purified) from extracts of Escherichia coli K-12, is a trimeric oligomer composed of identical or nearly identical subunits. The data include: (a) determining an overall average molecular weight of 64,700 for the native enzyme using the methods of sucrose density gradient centrifugation (64,000), gel filtration (62,500), polyacrylamide gel electrophoresis (68,000), and high speed meniscus depletion sedimentation equilibrium (66,000); (b) establishing by gel electrophoresis in sodium dodecyl sulfate that a single protein band of molecular weight =i 21,000 is obtained; (c) finding that cross-linking of the molecule with dimethyl suberimidate (or glutaraldehyde) followed by gel electrophoresis under denaturing conditions yields three distinct protein bands with integral molecular weights of 21,000, 42,000, and 64,000; ( d ) calculating a minimum molecular weight of 21,169 on the basis of amino acid analysis results which showed 3 histidine or 3 tryptophan residues/mol of 63,000; (e) finding with specific reagents the numbers and kinds of peptides in a tryptic digest of the aldolase to be most consistent with three (rather than two or four) subunits; ( f ) detecting and quantitatively measuring 3 mol of COOH-terminal eucine/mol of enzyme during digestion with carboxypeptidase A; (g) establishing that 3 mol of [14C]pyruvate/ mol of enzyme are bound by reduction of the aldolaseketimine adduct with cyanoborohydride; and (h) resolving, after dissociation and reassociation of mixtures of native and chemically modified (with [14C]maleic anhydride) aldolase subunits, a set of four hybrid aldolase species. In this last experimental approach, the pattern of enzymatic and radioactivity found in the four hybrids is consistent with their being designated as N3, N2M, N M 2 , and Ma (N = native and M = maleylated subunits). Since all efforts (dansylation, carbamylation, and dinitrophenylation) to identify the NH2-terminal amino acid of E. coli 2-keto-4-hydroxyglutarate aldolase failed, it appears that this end of the polypeptide chains is blocked.