Mutations to a glycine loop in the catalytic site of human mitochondrial Lon changes its protease, peptidase and ATPase activities

Authors: Nina Kunová 1    Ľuboš Ambro 1    Vladimír Pevala 1    Gabriela Ondrovičová 1    Jana Bellová 1    Eva Kutejová 1,2    Jacob Bauer 1   
1 Ústav molekulárnej biológie, SAV, Bratislava, Slovensko    2 Ústav mikrobiológie, AVČR, Praha   
Year: 2014
Section: Cellular metabolism, physiology, molecular biology and genetics
Abstract No.: 1057
ISBN: 978-80-970712-6-4

Lon, also called protease La, is an ATP-dependent protease present in all kingdoms of life. It is involved in protein quality control and several regulatory processes. Eukaryotic Lon possesses three domains, an N-terminal domain, an ATPase domain and a proteolytic domain. It requires ATP hydrolysis to digest larger, intact proteins, but can cleave small, fluorogenic peptides such as Glu-Ala-Ala-Phe-MNA by only binding, but not hydrolyzing, ATP. Both ATPase and peptidase activities can be stimulated by the binding of a larger protein substrate, such as β-casein. To better understand its mechanism of action, we have prepared several single or double point mutants of four conserved residues of human mitochondrial Lon (G893, G894, T880, W770) and studied their ATPase, protease and peptidase activities. Our results show that mutations to Gly894 enhance its basal ATPase activity but do not change its β-casein-stimulated activity. The loop containing Gly893 and Gly894, which flanks Lon’s proteolytic active site, therefore appears to be involved in the conformational change that occurs upon substrate binding. Furthermore, mutations to Trp770 have the same general effects on the ATPase activity as mutations to Gly893, indicating that Trp770 is involved in ATPase stimulation. We have also established that this loop does not need to move in order to cleave small, fluorogenic peptides, but does move during the digestion of β-casein. Finally, we also noted that Lon’s ability to digest small peptides can be inhibited by moderate ATP concentrations.

The authors would like to thank Carolyn Suzuki of the Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey, USA, for the gift of the plasmids encoding human Lon and the human Lon S855A mutant used in this study. This research was funded by the Slovak Research and Development Agency (Grant APVV-0123-10) and by the Slovak Grant Agency (Grant VEGA 2/0122/11).