Aminoacylase I (ACYI) catalyzes the stereospecific hydrolysis of L-acylamino acids and is generally assumed to be involved in the final step of the degradation of intracellular N-acetylated proteins. Apart from its crucial functions in intracellular amino acid metabolism, ACYI also has substantial commercial importance for the optical resolution of N-acylated DL-amino acids. As a zinc-dependent enzyme, ACYI is quite stable against heat-induced denaturation and can be regarded as a thermostable enzyme with an optimal temperature for activity of approximately 65 degrees C. In this research, the sequential events in ACYI thermal denaturation were investigated by a combination of spectroscopic methods and related resolution-enhancing techniques. Interestingly, the results from fluorescence and infrared (IR) spectroscopy clearly indicated that a pretransitional stage existed at temperatures from 50 degrees C to 66 degrees C. The thermal unfolding of ACYI might be a three-state process involving an aggregation-prone intermediate appearing at approximately 68 degrees C. The pretransitional structural changes involved the partial unfolding of the solvent-exposed beta-sheet structures and the transformation of about half of the Class I Trp fluorophores to Class II. Our results also suggested that the usage of resolution-enhancing techniques could provide valuable information of the step-wise unfolding of proteins. Study holds ProTherm entries: 23582, 23583, 23584, 23585, 23586, 23587, 23588, 23589 Extra Details: amino acid metabolism; thermostable; optimal temperature activity; structural changes
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:54 p.m.
|Number of data points||8|
|Assays/Quantities/Protocols||Experimental Assay: Tm prot_conc:1 mg/mL ; Experimental Assay: Tm buffers:Deuterated phosphate saline: 100 mM, prot_conc:50 mg/mL, pH:8.0 ; Experimental Assay: Tm prot_conc:2 microM|
|Libraries||Mutations for sequence MASKGREGEHPSVTLFRQYLRIRTVQPEPDYGAAVAFLEERARQLGLGCQKVEVVPGHVVTVLTWPGTNPTLSSILLNSHTDVVPVFKEHWSHDPFEGFKDADGYIYGRGAQDMKCVSIQYLEAVRRLKVEGHHFPRTIHMTFVPDEEVGGHQGMELFVKRPEFQALRAGFALDEGLASPTDAFTVFYSERSPWWLRVTSTGKPGHGSRFIEDTAAEKLHKVINSILAFREKEKQRLQSNQLKPGAVTSVNLTMLEGGVAYNVVPATMSACFDFRVAPDVDLKAFEEQLQSWCQAAGEGVTFEFVQKWMETQVTSTDDSDPWWAAFSGVFKDMKLALELEICPASTDARYIRAAGVPALGFSPMNHTPVLLHDHDERLHEAVFLRGVDIYTQLLSALASVPALPSES|