Proline:tRNA ligase from Thermus aquaticus was purified to homogeneity and characterized. Its molecular weight was found to be 127 000, consisting of two identical subunits. It catalysed the prolylation of tRNAPro from Escherichia coli with a bell-shaped pH dependence peaking at about pH 7 and exhibited extreme thermostability. The Vm/Km ratios of steady-state kinetics for proline and ATP as well as tRNAPro were not extensively diminished even at 85 degrees C, but prolylation became insignificant at 90 degrees C. Since the melting of tRNAPro was in progress, yet incomplete, at 85 degrees C, these findings suggest that some threshold level of conformational integrity of tRNAPro, rather than the entire unmelted conformation of the molecule, is essential to effective recognition by the proline:tRNA ligase.

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