Tryptophanyl Transfer Ribonucleic Acid Synthetase of Escherichia coli: II. MOLECULAR WEIGHT, SUBUNIT STRUCTURE, SULFHYDRYL CONTENT, AND SUBSTRATE-BINDING PROPERTIES

Abstract

Abstract The molecular weight of tryptophanyl-tRNA synthetase of Escherichia coli is 74,000 by Sephadex gel filtration and 81,000 by sucrose gradient centrifugation. Both sodium dodecyl sulfate polyacrylamide gel electrophoresis and agarose gel filtration in 8 m urea reveal subunits of 37,000 in molecular weight. In addition only one protein band is observed after polyacrylamide disc gel electrophoresis in the presence if 8 m urea. By these criteria the enzyme is a dimer composed of subunits identical in mass and charge. The enzyme catalyzes synthesis of the unusual product, tryptophanyl adenosine triphosphate ester (tryptophanyl-ATP). In attempts to form complexes with tryptophan, ATP, and enzyme we recovered the enzyme in a complex with tryptophanyl-ATP, 1.7 to 1.8 moles of tryptophanyl-ATP per mole of active enzyme. A symmetric dimer model was supported by studies with 5,5'-dithiobis(2-nitrobenzoic acid). The reagent detected two sulfhydryl groups per mole of enzyme, and both thiols reacted with the same first order rate constant. The sulfhydryl groups were essential for enzymatic activity and were protected from 5,5'-dithiobis(2-nitrobenzoic acid) by tryptophan and ATP together but not individually. A complex of tryptophanyl-tRNA-enzyme (0.9:1.0) is isolable by sucrose gradient centrifugation.