Abstract

To reveal the basis of the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of the enzyme and of its mesophilic counterpart from Escherichia coli has been studied. The urea-induced equilibrium unfolding of T. thermophilus and E. coli IPMDHs at 27 degreesC was monitored by measuring the changes in far-UV CD, intrinsic fluorescence, anilinonaphthalenesulfonic acid (ANS) binding, and catalytic activity in the presence of nonionic detergent Tween 20. For both enzymes, the spectral methods revealed a biphasic unfolding transition. The first transition was protein concentration-independent, whereas the second was protein concentration-dependent for both enzymes. The observation suggested a three-state unfolding mechanism with a dimeric intermediate. However, the intermediates of the E. coli and the T. thermophilus IPMDHs seemed to be different from each other. The intermediate of the E. coli IPMDH lost its secondary and tertiary structure more than that of the thermophilic enzyme. E. coli IPMDH lost enzymatic activity through the transition from the native to the intermediate state, though the intermediate of the T. thermophilus enzyme was still active. The unfolding process of E. coli IPMDH can be explained by a sequential unfolding of individual folding domains, while there is only a small structural perturbation in the intermediate of T. thermophilus IPMDH. The higher thermal stability of T. thermophilus IPMDH can be attributed to the increase in the extent of interaction inside the first domain which unfolded prior to the unfolding of the whole molecular structure in E. coli IPMDH. Study holds ProTherm entries: 5731, 5732, 5733, 5734 Extra Details: the transition is from native to intermediate

Submission Details

ID: ToYM75QC4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:30 p.m.

Version: 1

Publication Details

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