Abstract
AbstractThe glyoxalase system is a ubiquitously expressed enzyme system with narrow substrate specificity and is responsible for the detoxification of harmful methylglyoxal (MG), a spontaneous by-product of energy metabolism. Glyoxalase 1 (Glo1) is the first and therefore rate limiting enzyme of this protective system. In this study we were able to show that a phosphorylation of threonine-107 in the Glo1 protein, mediated by Ca2+/Calmodulin-dependent Kinase II delta (CamKIIδ), is associated with elevated catalytic efficiency of Glo1. In fact, Michaelis-Menten kinetics of Glo1 mutants revealed that a permanent phosphorylation of Glo1 was associated with increased Vmax(1.23 µmol/min/mg) and decreased Km(0.19 mM HTA), whereas the non-phosphorylatable Glo1 showed significantly lower Vmax(0.66 µmol/min/mg) and increased Km(0.31 mM HTA). This was also confirmed with human recombinant Glo1 (Vmax(Glo1phos) = 999 µmol/min/mg; Km(Glo1phos) = 0.09 mM HTA vs. Vmax(Glo1red) = 497 µmol/min/mg; Km(Glo1red) = 0.12 mM HTA). Additionally, proteasomal degradation of non-phosphorylated Glo1 via ubiquitination occurred more rapidly as compared to native Glo1. The absence of the responsible kinase CamKIIδ was associated with poor MG detoxification capacity and decreased protein content of Glo1 in a murine CamKIIδ knock-out model. Furthermore, this regulatory mechanism is also related to an altered Glo1 status in cancer, diabetes and during aging. In summary, phosphorylation of threonine-107 in the Glo1 protein by CamKIIδ is a quick and precise mechanism regulating Glo1 activity.
Publisher
Cold Spring Harbor Laboratory