Aspartic acid 320 is required for optimal activity of rat pancreatic cholesterol esterase.

摘要: The acidic amino acid residue required for the catalytic activity of rat pancreatic cholesterol esterase has been identified in this study by sequence comparison with other serine esterases and site-directed mutagenesis experiments. studies 3 residues homologous domains between esterase, acetylcholinesterase, cholinesterase, Geotrichum candida lipase that may potentially be these proteins. role Glu78, Asp79, Asp320 was then addressed expression cDNA. Results showed replacement Glu78 or Asp79 alanine no effect on ability to hydrolyze artificial water-soluble substrate p-nitrophenyl butyrate. In contrast, Asp320-->Ala320 substitution abolished enzyme esterase. specific requirement optimal demonstrated aspartic glutamic acid, thus retaining charge unit at position. Asp320-->Glu320 resulted an displayed normal interaction bile salt. However, mutagenized protein reduced approximately 50%. These results strongly suggested 320 is important component triad instead different from members gene family.