Dissecting the pretransitional conformational changes in aminoacylase I thermal denaturation

¹ Tsinghua University

^* To whom correspondence should be addressed. E-mail: ybyan{at}tsinghua.edu.cn.

Submitted on July 19, 2006
Revised on September 7, 2006
Accepted on 11 October 2006

	Abstract

Aminoacylase I (ACYI) catalyzes the stereo-specific 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 about 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 about 68°C. The pretransitional structural changes involved the partial unfolding of the solvent-exposed -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.

Key Words: Aminoacylase I, Fourier transform infrared spectroscopy, intrinsic fluorescence, resolution-enhancing technique, step-wise thermal unfolding, thermal denaturation