Quaternary Structure and Catalytic Functioning of l-Phenylalanine: tRNA Ligase of Escherichia coli K10

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.