Converting ALDH1 from being inhibited to being activated by Mg 2+ ions

Abstract

Cytosolic and mitochondrial aldehyde dehydrogenase share 70% sequence identity and have essentially the same 3-dimensional structure. Though the enzyme catalyzed reaction is presumed to be identical, the rate-limiting steps are not. For the cytosolic enzyme it is coenzyme dissociation, while for the mitochondrial enzyme it is the hydrolysis of the acyl intermediate formed after the hydride ion is transferred from the thiohemi-acetal to NAD. Mg2+ ions were found to enhance the deacylation step while retarding the rate of NADH dissociation. Thus, at Vm conditions, cytosolic ALDH1 is inhibited by the ions, while the mitochondrial isozyme is activated. Using a random mutational approach it was possible to select ALDH1 variants that were activated by Mg2+ ions. A point mutation producing T244S was responsible for the change in Mg2+-ion effect on the mutants. The T244S variant constructed by site-directed mutagenesis had a lower specific activity than the native enzyme. The rate-limiting step for the variant became deacylation like the mitochondrial isoform. Interestingly, the mitochondrial enzyme possesses a threonine at position 244 yet it is activated by the metal ion. Its coenzyme can sample two conformations such that the nicotinamide ring is found in two locations. The 3-dimensional structure of the binary complex shows that T244 is located near the nicotinamide ring that is in the hydride transfer conformation. Though the structure of the T244S variant has not been solved, it appears that the loss of possible hydrophobic interaction of the methyl group may change the nicotinamide ring conformation that caused the rate-limiting step to change. This work was supported in part by NIH Grant AA05812.