TRUNCATIONS AT THE NH2 TERMINUS OF RHODANESE DESTABILIZE THE ENZYME AND DECREASE ITS HETEROLOGOUS EXPRESSION

Rhodanese mutants containing sequential NH2-terminal deletions were co nstructed to test the distinct contributions of this region of the pro tein to expression, folding, and stability. The results indicate that the first 11 residues are nonessential for folding to the active confo rmation, but they are necessary for attaining an active, stable struct ure when expressed in Escherichia coli, Rhodanese species with up to 9 residues deleted were expressed and purified. Kinetic parameters for the mutants were similar to those of the full-length enzyme. Compared with shorter truncations, mutants missing 7 or 9 residues were (a) inc reasingly inactivated by urea denaturation, (b) more susceptible to in activation by dithiothreitol, (c) less able to be reactivated, and (d) less rapidly inactivated by incubation at 37 degrees C, Immunoprecipi tation showed that mutants lacking 10-23 NH2-terminal amino acids were expressed as inactive species of the expected size but were rapidly e liminated. Cell-free transcription/translation at 37 degrees C showed mutants deleted through residue 9 were enzymatically active, but they were inactive when deleted further, just as in vivo, However, at 30 de grees C in vitro, both Delta 1-10 and Delta 1-11 showed considerable a ctivity. Truncations in the NH2 terminus affect the chemical stability of the distantly located active site. Residues Ser-11 through Gly-22, which form the NH2-proximal alpha-helix, contribute to folding to an active conformation, to resisting degradation during heterologous expr ession, and to chemical stability in vitro.