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Dissecting the Pretransitional Conformational Changes in Aminoacylase I Thermal Denaturation

State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China

Correspondence: Address reprint requests to Dr. Yong-Bin Yan, Dept. of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China. Tel.: 86-10-6278-3477; Fax: 86-10-6277-1597; E-mail: ybyan{at}tsinghua.edu.cn.

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 65°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°C to 66°C. The thermal unfolding of ACYI might be a three-state process involving an aggregation-prone intermediate appearing at 68°C. The pretransitional structural changes involved the partial unfolding of the solvent-exposed ß-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.