Abstract
Cyclooxygenase catalysis by prostaglandin H synthase-1 and -2 (PGHS-1 and -2) requires activation of the normally latent enzyme by peroxide-dependent generation of a free radical at Tyr-385 (PGHS-1 numbering) in the cyclooxygenase active site; the Tyr-385 radical has also been linked to self-inactivation processes that impose an ultimate limit on cyclooxygenase catalysis. Cyclooxygenase activation is more resistant to suppression by cytosolic glutathione peroxidase in PGHS-2 than in PGHS-1. This differential response to peroxide scavenging enzymes provides a basis for the differential catalytic regulation of the two PGHS isoforms observed in vivo. We sought to identify structural differences between the isoforms, which could account for the differential cyclooxygenase activation, and used site-directed mutagenesis of recombinant human PGHS-2 to focus on one heme-vicinity residue that diverges between the two isoforms, Thr-383, and an adjacent residue that is conserved between the isoforms, Asn-382. Substitutions of Thr-383 (histidine in most PGHS-1) with histidine or aspartate decreased cyclooxygenase activation efficiency by about 40%, with little effect on cyclooxygenase specific activity or self-inactivation. Substitutions of Asn-382 with alanine, aspartate, or leucine had little effect on the cyclooxygenase specific activity or activation efficiency but almost doubled the cyclooxygenase catalytic output before self-inactivation. Asn-382 and Thr-383 mutations did not appreciably alter the Km value for arachidonate, the cyclooxygenase product profile, or the Tyr-385 radical spectroscopic characteristics, confirming the structural integrity of the cyclooxygenase site. The side chain structures of Asn-382 and Thr-383 in PGHS-2 thus selectively influence two important aspects of cyclooxygenase catalytic regulation: activation by peroxide and self-inactivation.
Highlights
This suggests that the constraints on side chain structure at residue 383 are greater in PGHS-2 than in PGHS-1, and is consistent with the observation that both the T383H and T383D mutations in PGHS-2 decreased the cyclooxygenase activation efficiency toward that seen in PGHS-1 (Table III)
Cyclooxygenase velocities were predicted in simulated reactions of a fixed amount of PGHS with various levels of cytosolic glutathione peroxidase (cGPx), with the resulting cyclooxygenase velocities normalized to the control value and plotted as a function of the ratio of cGPx units to cyclooxygenase units, as was done for the actual enzymes in Fig. 4. cGPx titration slope values from Fig. 4 were 2.4 ϫ 10Ϫ3 for PGHS-2, 15.4 ϫ 10Ϫ3 for PGHS-1, and near 5.3 ϫ 10Ϫ3 for the Thr-383 mutants
Summary—The results identify the Thr-383 side chain structure as a selective determinant of cyclooxygenase feedback activation efficiency and identify the side chain structure of Asn-382 as a selective determinant of cyclooxygenase selfinactivation characteristics
Summary
Materials—Arachidonic acid was purchased from NuChek Preps, Inc. Heme, phenol, TMPD, glutathione, glutathione reductase, and cGPx were from Sigma. Tween 20 was purchased from Pierce Chemical Co. Recombinant wild-type and mutant PGHS-2 were expressed in Sf9 cells using a baculovirus vector as described below and reconstituted with excess heme before use [12]. The supernatant liquid containing solubilized PGHS-2 was clarified by passage through a 0.2-m filter and divided into aliquots for storage at Ϫ70 °C This procedure results in at least a 10-fold purification of the cyclooxygenase activity from the Sf9 cell homogenate and provides a good yield of recombinant PGHS-2 suitable for reliable characterization of enzymatic activity, susceptibility to suppression by cGPx and, after buffer exchange to lower the phenol concentration, EPR spectroscopy. Aliquots of solubilized recombinant wild-type or mutant PGHS-2 (ϳ40 units) were added and after 30 s the reaction was stopped by addition of 0.75 ml of ice-cold ethyl ether-methanol-2 M citric acid (30:4:1). For HPLC analysis, 20 l of each extract (2000 –5000 cpm) was injected on a 0.46 ϫ 15 cm Varian Microsorb-MV-
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