Possible Structure and Function of the Extra C-Terminal Sequence of Pyruvate Kinase from Bacillus stearothermophilus

Abstract

The pyruvate kinases from Genus Bacillus and a few other bacteria have an extra C-terminal sequence with a phosphoenolpyruvate binding motif composed of about 110 amino acids. To elucidate the possible structure and function of this sequence, the enzyme lacking the sequence was prepared and characterized. The N-terminal sequences of the peptides, which were found only in the lysylendopeptidase digest of the wild enzyme and not in that of the truncated enzyme, were determined. All the determined sequences were found in the extra C-terminal sequence deduced from the DNA sequence. The truncated enzyme showed decreased affinity for phosphoenolpyruvate and the allosteric effector ribose 5-phosphate, and had a reduced thermostability. Other properties, such as tetrameric structure, specific activity, and allosteric characteristics were unchanged. A comparison of the CD spectra of the truncated enzyme and the recombinant enzyme indicated that the structure of the C-terminal sequence should be rich in beta-sheet. These findings suggest that the sequence actually exists and that it may form a steady domain interacting with the A-domain and C-domain, which are the catalytic domain and allosteric effector binding domain, respectively.