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Int J Biol Macromol. 2013 Jul;58:66-72. doi: 10.1016/j.ijbiomac.2013.03.038. Epub 2013 Mar 31.

Thermal inactivation and conformational lock studies on horse liver alcohol dehydrogenase: structural mechanism.

Author information

1
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.

Abstract

Horse liver alcohol dehydrogenase (HLADH) is a two subunits metal enzyme that has two catalytic sites and two coenzyme domains for each subunit. These subunits are connected together by coenzyme domains. In this study, we investigated the number and sequences of residues that participated in interface locks of HLADH. For this purpose, the kinetics of thermal inactivation of HLADH were studied in a 50 mM pyrophosphate buffer, pH 8.8, using ethanol as a substrate and NAD(+) as a cofactor. The temperature range was between 46°C and 55°C and the conformational lock was developed based on the Poltorak theory and analysis of the curves was done by the conformational lock method for oligomeric enzymes. The conformational lock number of HLADH was 2 when calculated experimentally. The results were confirmed by the Ligplot program computations. Using computational method it was shown that there are two patches binding sites at the interface and they spread over two regions of each chain. In this study we also proposed a thermal denaturation mechanism for HLADH by using different techniques such as UV-Vis fluorescence and circular dichroism (CD) spectroscopy and dynamic light scattering (DLS). The subunits are dissociated and several intermediates appeared during inactivation through increasing the temperature. DLS measurement was performed to study the changes in hydrodynamic radius during thermal inactivation. The three distinct zones that were shown by DLS were also confirmed by fluorescence and CD techniques.

PMID:
23548863
DOI:
10.1016/j.ijbiomac.2013.03.038
[Indexed for MEDLINE]

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