Tryptophanase from Aeromonas liquefaciens. Purification molecular weight and some chemical, catalytic and immunochemical properties

Abstract

Tryptophanase from Aeromonas liquefaciens has been purified to homogeniety by employing a heat treatment, (NH 4) 2SO 4 fractionations and hydroxyapatite chromatography. The enzyme binds four moles of pyridoxal 5′-phosphate per mole of enzyme, has s 0 2-, w value of 9.78 S and a mol. wt of 216 000. These properties and the general catalytic properties of the enzyme are similar to other tryptophanase molecules. The enzyme requires pyridoxal 5′-phosphate ( K m 2.09·10 −6 M), NH 4 + or K + for catalytic activity and besides using tryptophan ( K m 2.21·10 −4 M), the enzyme can also utilize l-serine, l-cysteine and S- methyl- l-cysteine in catalyzing α,β-elimination and β-replacement reactions. Anthranilic acid ( K i 3.0·10 −4 M) alanine ( K i 1.31·10 −3 M), and phenylalanine ( K i 7.79·10 −3 M) are competitive inhibitors of the tryptophanase reaction. Immunochemical studies indicate that the A. liquefaciens enzyme has some common antigenic determinant sites with the tryptophanases from Escherichia coli, Bacillus alvei, a marine vibrio (K-7), Paracolobactrum coliforme, Micrococcus aerogenes, and Sphaerophorus funduliformis. Each tryptophanase studied has a distinct amino acid composition, but as a group they are characterized by a fairly high content (50–56%) of non-polar amino acids and a very similar total basic amino acid content. The A. liquefaciens enzyme has the highest non-polar and lowest acidic amino acid content of the group, and may be expected to have an electrophoretic mobility significantly different from the other molecules.