Properties and subunit structure of the B component of Pseudomonas putida tryptophan synthetase

Abstract

The purified B component of Pseudomonas putida tryptophan synthetase has absorption maxima at 278, 332, and 415 nm in potassium phosphate buffer at pH 7.0. After borohydride reduction, with the cofactor covalently attached to the polypeptide chain, the protein loses the maxima at 332 and 415 nm, but acquires a new absorption band at 320 nm. The K m for the binding of pyridoxal-5′-phosphate to the enzyme was found to be 5 × 10 −7 m. One of the substrates in the indole to tryptophan reaction, l-serine, has a marked effect on the heat stability of the holoenzyme. The molecular weight determined by acrylamide gel electrophoresis and by ultracentrifugation is 86,000 ± 2,000. Upon treatment with detergent, performic acid, or urea, two identical subunits half the size of the native enzyme are found. Two alanine residues are released by carboxypeptidase A digestion of performic acid-oxidized B protein. No strong aggregates are formed between E. coli tryptophan synthetase A protein and P. putida B protein, and no hybrid B protein is detectable after dissociation of a mixture of E. coli and P. putida B proteins in urea and reaggregation in potassium phosphate buffer.