L-Phenylalanine : tRNA ligase was inactivated and dissociated into subunits by exposure to guanidinium chloride, a pH of 2 or p-chloromercuribenzoate. After removal of the denaturant the enzyme quaternary structure was restored. In the case of acid denaturation it was shown that the specific activities of the restored and native enzyme were identical. At pH 2 and in guanidinium chloride dissociation proceeded directly to monomers while preliminary evidence was obtained that modification with p-chloromercuribenzoate at pH 7.5 was accompanied by dissociation into dimers. After exposure to pH 8.9 the modified dimers subsequently dissociated into monomers. The quaternary structures were differentiated by polyacrylamide gel electrophoresis, gel chromatography and kinetic analysis.Preparations of considerable purity were obtained for both monomers by polyacrylamide gel electrophoresis or sucrose gradient centrifugation; however, complete separation was accomplished only by gel chromatography of guanidinium-chloride-dissociated protein. Separated subunits had no detectable enzymic activity nor the ability to bind tRNAPhe and L-phenylalanine. However, when recombined, the enzymic properties were restored.An effect of the substrates and of magnesium was observed as a protection against the dissociation and inactivation of the enzyme when exposed to p-chloromercuribenzoate. An effect of magnesium was also observed during reassociation of the enzyme from pH-2-generated monomers. The rate of the reactivation was considerably enhanced by the presence of the cation, probably via a rapid formation of dimeric subunits.The results provide evidence that the integral quaternary structure of L-phenylalanine : tRNA ligase is a prerequisite for the functioning of the active site.Amino acid:tRNA ligases exhibit a variety of different subunit structures [l ] (and references therein). Among those which are more complicated is the L-phenylalanine-specific enzyme of Escherichia coli with an a2B2 type of subunit structure [2]. Despite its high molecular weight of 270000 the enzyme has recently been shown to reassociate from urea-dissociated monomers [3], and thereby regain part of its catalytic activity. The purpose of the present paper is to extend these studies to other conditions which gently lead to dissociation into monomers and subsequent reassociation to fully active enzyme. The subunits were isolated and investigated for enzyme and substrate binding properties at conditions which would favour reassociation to the native enzyme.Enzyme. L-Phenylalanine: tRNA Ligase (EC 6.1.1.20).Eur. J. Biochem. 56 (1975) Moreover, the effect of substrates and magnesium on the dissociation and reassociation behaviour was investigated. Evidence is provided that the catalytic functioning and the intact quaternary structure are intimately related properties of the enzyme.
MATERIALS AND METHODSL-Phenylalanine : tRNA ligase was isolated from Escherichia coli K10 in the presence of phenylmethylsulfonyl fluoride as described previously [2,3] (and referen...
