Kinetic Isotope Effect of Prostaglandin H Synthase Exhibits Inverted Temperature Dependence

Gang Wu,Richard Kulmacz,Ah-Lim Tsai

doi:10.3390/catal4020174

Abstract

Conversion of arachidonic acid to prostaglandin G2/H2 catalyzed by prostaglandin H synthase (PGHS) is proposed to involve initial transfer of the C13 pro-(S) hydrogen atom from arachidonate to the Tyr385 radical in PGHS, followed by insertion of two oxygen molecules and several chemical bond rearrangements. The initial hydrogen-transfer was recently concluded to be a rate-limiting step in cyclooxygenase catalysis based on the observed intrinsic deuterium kinetic isotope effect values (Dkcat). In the present study, we have found that Dkcat values of both PGHS-1 and -2 show an unusual increase with temperatures in the range of 288–310 K, exhibiting an inverted temperature dependence. The value of lnDkcat, however, decreased linearly with 1/T, consistent with a typical Arrhenius relationship.

Highlights

Prostaglandin H synthase (PGHS) catalyzes the biosynthesis of prostaglandin H2 (PGH2), which is the precursor of all the prostaglandins, thromboxanes and prostacyclins [1,2]
The cyclooxygenase reaction starts with the abstraction of the C13 pro-(S) hydrogen of arachidonic acid (AA) by a tyrosyl radical located on Tyr385 in PGHS-1 (Tyr371 in PGHS-2)
We found that the activation energies for PGHS-1 or -2 with 10,10,13,13-d4-AA

Summary

Introduction

Prostaglandin H synthase (PGHS) catalyzes the biosynthesis of prostaglandin H2 (PGH2), which is the precursor of all the prostaglandins, thromboxanes and prostacyclins [1,2]. PGHS reacts with peroxide at its peroxidase site to generate Intermediate I, which contains a ferryl heme and Catalysts 2014, 4 a proto-porphyrin radical; subsequent intra-molecular electron transfer from an adjacent tyrosine residue to the ferryl heme yields Intermediate II and a tyrosine radical [3,4,5] This tyrosyl radical is the starting point of cyclooxygenase catalysis, which transforms arachidonic acid (AA) to prostaglandin. Observations of isotope enrichment in the unreacted substrate after PGHS incubations with tritium-substituted 8,11,14-eicosatrienoic acid indicated that the initial hydrogen abstraction from the substrate (step 1, Scheme 1) is rate-limiting in PGHS cyclooxygenase catalysis [9]. Most theoretical models based on transition state theory or hydrogen tunneling predict smaller activation energy for hydrogen than deuterium and a reciprocal correlation between KIE and temperature, called a normal temperature dependence [21,22]. There are only a few reported examples of KIE values increasing with temperature in chemical reactions [23,24,25,26,27] and enzymatic catalyses [16,28]

Results and Discussion

Materials

Measurement of Non-Competitive Kinetic Isotope Effect