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J. Biol. Chem., Vol. 279, Issue 31, 32093-32099, July 30, 2004

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Effect of pH on the Stability and Structure of Yeast Hexokinase A

ACIDIC AMINO ACID RESIDUES IN THE CLEFT REGION ARE CRITICAL FOR THE OPENING AND THE CLOSING OF THE STRUCTURE^*

D. Prasanna Kumar, Ashutosh Tiwari, and Rajiv Bhat

From the Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110 067, India

pH and salts have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. In this work, calorimetry, CD, and fluorescence studies have been carried out to understand the pH-dependent conformational changes of the two-domain protein yeast hexokinase A. In conjunction with the crystal structural data available, the present results have enabled the complete characterization and analysis of the pH-dependent conformational changes of the enzyme that have strong implications in understanding its structure-function relationship. The calorimetric profiles show a single thermal transition in the acidic pH range, whereas two independent transitions were observed in the alkaline pH range, suggesting the structural merger of the domains at the acidic pH. Comparison of the thermal transitions at pH 8.5 studied by different techniques suggests that the first transition corresponds to the smaller domain, and the second transition corresponds to the larger domain. The acid-denatured state of hexokinase A has high secondary structure content with little or no tertiary interactions and binds to the hydrophobic dye 8-anilinonaphthalene-1-sulfonic acid, suggesting that it is a molten globule-like state, whereas the alkali-denatured state is less structured than the acid-denatured state but more structured than the urea-denatured state, suggestive of a premolten globule-like state. Structural analysis using the published hexokinase B structure as well as the hexokinase A structure with the revised amino acid sequence in conjunction with the results obtained by us suggests that the ionization state of the acidic residues at the active site could regulate domain movements that are responsible for the opening and the closure of the cleft between the two domains and in turn affect the structure and function of the enzyme.

Received for publication, December 9, 2003 , and in revised form, April 16, 2004.

^* Use of the VP-DSC calorimeter was supported by the Department of Science and Technology, Government of India, under the Intensification of Research in High Priority Areas. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Neurology, University of Massachusetts Medical School, Worcester, MA 01655.

To whom correspondence should be addressed. Tel.: 91-11-26704086; Fax: 91-11-26717586; E-mail: rajivbhat{at}mail.jnu.ac.in.

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