Mutant of Human 12/15-lipoxygenase That Has Been Implicated in Coronary Artery Disease

Lipoxygenases (LOXs) have been implicated in cardiovascular disease. A rare single nucleotide polymorphism causing T560M exchange has recently been described and this mutation leads to a near null-variant of the enzyme encoded for by the ALOX15 gene. When we inspected the 3D-structure of the rabbit ortholog we localized T560 outside the active site and identified a hydrogen bridge between its side chain and Q294. This interaction is part of a complex hydrogen bond network, which appears to be conserved in other mammalian LOXs. Q294 and N287 are key amino acids in this network and we hypothesized that disturbance of this hydrogen bond system causes the low activity of the T560M mutant. To test this hypothesis we first mutated T560 to amino acids not capable of forming side chain hydrogen bridges (T560M, T560A) and obtained enzyme variants with strongly reduced catalytic activity. In contrast, enzymatic activity was retained after T560S exchange. Enzyme variants with strongly reduced activity were also obtained when we mutated Q294 (binding partner of T560) and N287 (binding partner of Q294 and M418) to L. Basic kinetic characterization of the T560M mutant indicated that the enzyme lacks a kinetic lag-phase but is rapidly inactivated. This data suggests that the low catalytic efficiency of the naturally occurring T560M mutant is caused by alterations of a hydrogen bond network interconnecting this residue with active site constituents. Disturbance of this bonding network increases the susceptibility of the enzyme for suicidal inactivation.