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Journal of Biochemistry 2007 142(2):247-255; doi:10.1093/jb/mvm126
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© 2007 The Japanese Biochemical Society.

Different Stabilities of Two AMP-forming Acetyl-CoA Synthetases from Phycomyces blakesleeanus Expressed under Different Environmental Conditions

Sergio de Cima, Javier Rúa, Pilar del Valle, Félix Busto, Alberto Baroja-Mazo{dagger} and Dolores de Arriaga*

Departamento de Biología Molecular, Universidad de León, Campus de Vegazana, 24007 León, Spain

*To whom correspondence should be addressed. Tel: +34 987 291 229, Fax: +34 987 291 226, E-mail: dolores.arriaga{at}unileon.es

Received May 17, 2007; Accepted June 18, 2007


  Abstract

The stability of acetyl-CoA synthetases (ACS1 and ACS2) from P. blakesleeanus against temperature, urea and trypsin was studied and compared. Thermal inactivation of ACS1 was biphasic, while that of ACS2 was monophasic. The thermodynamic parameters calculated from the inactivation profiles show ACS2 to be a more thermostable enzyme than ACS1. The presence of ATP and Mg2+ exerted a protective effect on both enzymes, and led to a marked increase in the Ea, {Delta}H!=, {Delta}S!= and {Delta}G!= values. ACS2 is also much more stable against denaturation with urea; the estimates of {Delta}Gw (free energy change for protein unfolding at zero denaturant concentration) were 9.4 kJ mol–1 and 18.1 kJ mol–1 for ACS1 and ACS2, respectively. Finally, a half-life of 44.5 min for ACS2 versus the 21 min for ACS1 indicates that ACS2 is more stable than ACS1 against digestion by trypsin. These results seem to show that ACS2 is more rigid overall than ACS1, which may be essential for preserving its catalytic activity in the stress situation in which it is expressed.

Key Words: Acetyl-CoA synthetases, filamentous fungi, stability, thermal inactivation, urea denaturation

Abbreviations: ACS, acetyl-CoA synthetase

{dagger}Present address: Hospital Universitario Virgen de la Arrixaca, Unidad de Investigación, Cirugía Experimental, Murcia, Spain


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