The amino acid sequence of beta-galactosidase of Escherichia coli.

Abstract

The amino acid sequence of (3-galactosidase was determined. The protein contains 1021 amino acid residues in a single polypeptide chain. The subunit molecular weight calculated from the sequence is 116,248. The sequence determination, carried out mainly by conventional methods, was aided by complementation tests, by the use -of termination mutant strains, and by a new immunochemical method. The five residue sequence Thr-Pro-His-Pro-Ala appears twice within the polypeptide chain, but no other striking homologous features are evident.f3-Galactosidase (f3-D-galactoside galactohydrolase, EC 3.2.1.23) is specified by the first structural gene (lac Z) of the lac operon in Escherichia coli. Physical and chemical studies have shown that the protein is a tetramer of four identical, unusually long, polypeptide chains. Estimates of the size of the monomer have varied from about 1000 to 1200 amino acid residues; the value of 1170 has been assumed in the past (1, 2).Although the determination of t~he primary structure of ,Bgalactosidase was a major undertaking, it seemed warranted for a number of reasons. Sequence ianformation is important in order to correlate some of the extensive genetic data available on the Z gene with the protein, to investigate enzyme structure-function relationships, and to study the origin of this single protein and other proteins of the lac operon by examination for homology.The amino acid sequence of #-galactosidase has now been completed and is presented here.RESULTS AND DISCUSSION The amino acid sequence proposed for ,B-galactosidase is shown in Fig. 1. From the composition (Table 1) molecular weights of 116,248 for the monomer and 464,992 for the tetramer were calculated.The sequence was derived by studies of peptides obtained by cleavage of the protein with trypsin, chymotrypsin, and cyanogen bromide (CNBr). Structure determination was initiated by isolation of tryptic peptides (3,4) including the aminoand carboxyl-terminal fragments (5). Additional large peptides were obtained from a tryptic digest of ,B-galactosidase blocked at lysine residues with citraconic anhydride. Details of peptide isolation and sequence determination will be published elsewhere.Of the 24 unique peptides produced by cyanogen bromide treatment, 8 ranging in size from 2 to 15 residues were purified by standard techniques of paper electrophoresis and paper chromatography. The 16 larger peptides, containing 23 to 119 residues, were chromatographed at pH 5.0 on a O-carboxymethylcellulose column in 0.02 M ammonium acetate buffer containing 8 M urea and were eluted with a salt gradient (6). The elution position of these peptides can be seen in Fig. 2 in low yield which were not cleaved at certain methionine residues, or peptides derived by cleavage of the three aspartyl-prolyl bonds in fl-galactosidase. All peptides were purified further by gel filtration and, in some cases, by additional ion-exchange chromatography procedures (6). Criteria of purity included dansyl amino-terminal analysis, electrophoresis on 7.5% po...

On the evolution of beta-galactosidase.

“…The amino acid sequence of f3-galactosidase (fl-D-galactoside galactohydrolase, EC 3.2.1.23) of Escherichia coli has recently been determined (4). Each of its four identical polypeptide chains contains 1021 amino acid residues.…”

Reevaluation of the Mode of Action of Streptolydigin in Escherichia coli: Induction of Transcription Termination In Vivo

“…Synthesis of the N-terminal part of the ,-galactosidase polypeptide chain, the auto-a peptide fragment (60 to 70 amino acids), and of complete ,B-galactosidase (1,021 amino acids [10]) was measured in strain NF830 after inducton by isopropyl-/t-D-thiogalactoside. The kinetics of accumulation of the two activities were identical in the control culture (Fig.…”

Amino acid sequence of fibrolase, a direct‐acting fibrinolytic enzyme from agkistrodon contortrix contortrix venom

“…Following digestion at room temperature for 24 h, the sample was dialyzed against 50% acetic acid in Spectra/Por 3 (Spectrum Medical Industries, Inc., Los Angeles, California) dialysis tubing that had been boiled for 5 min in 0.1 M EDTA (pH 8.0) to reduce the pore size so that peptides of seven residues or more are retained (Fowler, 1978). The digest was dried in a Speed Vac and redissolved in a small volume of 50% acetic acid prior to the initial peptide separation using Bio-Gel P-30.…”