Thesis (M.Sc.)--Chulalongkorn University, 2000

The initial phase of this work is to compare essential amino acid residues in each isoform of CGTase from Bacillus circulans A11. The isoforms were purified by preparative gel electrophoresis. Modification with certain group-specific reagents and measurement of the loss in isoform activities were performed. It was found that amino acid residues which were essential for all 4 isoforms were histidine, tryptophan, tyrosine, and carboxylic amino acids. Different residues identified to be essential for each isoform were: isoforms 2 and 4, serine ; isoform 3, lysine. From substrate protection experiment, by adding 25 mM methy1-Beta-CD prior to modification, amino acid residues which were found to be at the active site of each isoform were histidine, tryptophan, tyrosine, and carboxylic amino acids. In addition to these residues, serine in isoform 2 and lysine in isoform 3 were also protected by the substrate suggesting their presence at the active site. The second phase of this work is to characterize and determine the number and position of essential histidine (s) at the active site of isoform 1. When inactivation by diethylpyrocarbonate (DEP) was performed at pH 6.0, 40ํC, the suitable concentration of DEP was 0.325 mM, and the suitable incubation time was 5 minutes. Inactivation kinetics of isoform 1 with DEP resulted in the second-order rate constant (kinactivation) of 29.5M-1s-1. The ratio of DEP to isoform 1 (in mole unit) wa 1 : 1. Moreover, it was found that methy1-beta-CD protects two histidine residues of isoform 1. When isoform 1 was digested by trypsin, peptides resulting from enzymatic cleavage were separated by HPLC. It was observed that peptides of interest were those with Rt = 11.348 and 40.934 minutes. For the peak eluting at 11.348 minutes, mass spectrometry reveals the size Mr of 5,723 Da and the N-terminal sequence was F A A K. While the peak eluting at 40.934 minutes, the size Mr of 2,540 Da was obtained and the N-terminal sequence was V I I D F A P N H T. When the data from peptide analysis was checked with the wequence of CGTase, it could be predicted that His-140 and His-327 were essential histidines in the active site of isoform 1.