A study of the threonyl adenylate complex with threonyl transfer ribonucleic acid synthetase and its reaction with hydroxylamine.

Threonyl transfer ribonucleic acid synthetase from Escherichio coli was purified 320-fold in 10% yield. The K, for the over-all reaction, in which threonyl-tRNA is formed, is 1O-4 M for ATP and 12 X 1OM6 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-PP; exchange reaction. Threonyl adenylate-enzyme complex was isolated, and, in the presence of MgC12 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”.