title: Mutational analysis of the autoprocessing site of subtilisin YaB-G124A.
abstract: The potential residue at the autoprocessing site for improving processing efficiency was evaluated from hydrolysis of 19 cleavage-site-mimicking octapeptides, VTTXQTVP (-4 to +4), by the mature subtilisin YaB and YaB-G124A mutants. Both enzymes cleaved the octapeptides mainly at two sites, X-Q (A-site) and Q-T (B-site), at varied preferences. Based on the results above, Met(-1) of YaB-G124A was mutated and, as expected, extracellular enzyme production increased with Gln or Ala replacement, but decreased with Ile or Asp substitution. Together with previous structural studies, our results suggest that autoprocessing is dependent on not only the primary structure, but also the peptide flexibility around the processing site. Cleavage at the B-site resulted in a novel YaB mutant lacking the N-terminus Gln 1, which led the mutant enzyme to less enzymatic activity by 80% and less thermal stability by 20 degrees C, perhaps due to its ligation to the high-affinity calcium ion.
authors.affiliation: Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan, Republic of China.
authors.forename: Yuh-Shin
authors.initials: YS
authors.lastname: Chang
authors.forename: Shwu-Huey
authors.initials: SH
authors.lastname: Liaw
authors.forename: Hui-Ching
authors.initials: HC
authors.lastname: Mei
authors.forename: Chih-Chieh
authors.initials: CC
authors.lastname: Hsu
authors.forename: Chi-Yu
authors.initials: CY
authors.lastname: Wu
authors.forename: Ying-Chieh
authors.initials: YC
authors.lastname: Tsai
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D010455:Peptides; D020860:Subtilisin