Dissecting the pretransitional conformational changes in aminoacylase I thermal denaturation.
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.
Su
Jing-Tan
JT
State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China.
Kim
Sung-Hye
SH
Yan
Yong-Bin
YB
D000581:Amidohydrolases / Q000737:chemistry* / Q000648:ultrastructure*; D003198:Computer Simulation; D006358:Hot Temperature; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation
D000581:Amidohydrolases; C019672:aminoacylase I