Lipoxygenases 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 three-dimensional structure of the rabbit ortholog, we localized Thr-560 outside the active site and identified a hydrogen bridge between its side chain and Gln-294. This interaction is part of a complex hydrogen bond network that appears to be conserved in other mammalian lipoxygenases. Gln-294 and Asn-287 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 Thr-560 to amino acids not capable of forming side chain hydrogen bridges (T560M and 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 Gln-294 (binding partner of Thr-560) and Asn-287 (binding partner of Gln-294 and Met-418) to Leu. Basic kinetic characterization of the T560M mutant indicated that the enzyme lacks a kinetic lag phase but is rapidly inactivated. These data suggest 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.