A Study of the Threonyl Adenylate Complex with Threonyl Transfer Ribonucleic Acid Synthetase and Its Reaction with Hydroxylamine

Threonyl transfer ribonucleic acid synthetase from Escherichia coli was purified 320-fold in 10% yield. The Km for the over-all reaction, in which threonyl-tRNA is formed, is 10-4 m for ATP and 12 x 10-6 m for threonine. The molecular weight is 117,000 ± 9% as determined by sucrose density gradient centrifugation. The enzyme readily catalyzes a threonine-dependent ATP-PPi exchange reaction. Threonyl adenylate-enzyme complex was isolated, and, in the presence of MgCl2 and tRNA, the amino acid was rapidly esterified to tRNA. At pH 7, the complex has a half-life of 29 min at 30° and 78 min at 10°. When threonyl adenylate-enzyme complex is treated with hydroxylamine, no threonine hydroxamate is formed and free threonine is the product, but if the complex is first treated with p-chloromercuribenzoate followed by hydroxylamine, threonine hydroxamate can be recovered in 50% yield. The phenylalanyl adenylate-enzyme complex was isolated, and it readily reacted with hydroxylamine to form phenylalanine hydroxamate. This complex has a half-life of 7 min at 30° and 35 min at 10°.