Prostaglandin H synthase: Resolved and unresolved mechanistic issues

The cyclooxygenase and peroxidase activities of prostaglandin H synthase (PGHS)-1 and -2 have complex kinetics, with the cyclooxygenase exhibiting feedback activation by product peroxide and irreversible self-inactivation, and the peroxidase undergoing an independent self-inactivation process. The mechanistic bases for these complex, non-linear steady-state kinetics have been gradually elucidated by a combination of structure/function, spectroscopic and transient kinetic analyses. It is now apparent that most aspects of PGHS-1 and -2 catalysis can be accounted for by a branched chain radical mechanism involving a classic heme-based peroxidase cycle and a radical-based cyclooxygenase cycle. The two cycles are linked by the Tyr385 radical, which originates from an oxidized peroxidase intermediate and begins the cyclooxygenase cycle by abstracting a hydrogen atom from the fatty acid substrate. Peroxidase cycle intermediates have been well characterized, and peroxidase self-inactivation has been kinetically linked to a damaging side reaction involving the oxyferryl heme oxidant in an intermediate that also contains the Tyr385 radical. The cyclooxygenase cycle intermediates are poorly characterized, with the exception of the Tyr385 radical and the initial arachidonate radical, which has a pentadiene structure involving C11-C15 of the fatty acid. Oxygen isotope effect studies suggest that formation of the arachidonate radical is reversible, a conclusion consistent with electron paramagnetic resonance spectroscopic observations, radical trapping by NO, and thermodynamic calculations, although moderate isotope selectivity was found for the H-abstraction step as well. Reaction with peroxide also produces an alternate radical at Tyr504 that is linked to cyclooxygenase activation efficiency and may serve as a reservoir of oxidizing equivalent. The interconversions among radicals on Tyr385, on Tyr504, and on arachidonate, and their relationships to regulation and inactivation of the cyclooxygenase, are still under active investigation for both PGHS isozymes.     

The molecular basis of color vision in colorful fish: Four Long Wave-Sensitive (LWS) opsins in guppies (<Emphasis Type="Italic">Poecilia reticulata</Emphasis>) are defined by amino acid substitutions at key functional sites

Background  

Comparisons of functionally important changes at the molecular level in model systems have identified key adaptations driving isolation and speciation. In cichlids, for example, long wavelength-sensitive (LWS) opsins appear to play a role in mate choice and male color variation within and among species. To test the hypothesis that the evolution of elaborate coloration in male guppies (Poecilia reticulata) is also associated with opsin gene diversity, we sequenced long wavelength-sensitive (LWS) opsin genes in six species of the family Poeciliidae.     

Prostaglandin H synthase. Effects of peroxidase cosubstrates on cyclooxygenase velocity

Many cosubstrates for the peroxidase activity of prostaglandin H synthase-1 (PGHS-1) have been reported to produce a large (2-7-fold) increase in the cyclooxygenase velocity in addition to a substantial increase in the number of cyclooxygenase catalytic turnovers. The large stimulation of cyclooxygenase velocity has become an important criterion for evaluation of putative PGHS reaction mechanisms. This criterion has been a major weakness of branched-chain tyrosyl radical mechanisms, which correctly predict many other cyclooxygenase characteristics. Our computer simulations based on a branched-chain mechanism indicated that the uncorrected oxygen electrode signals commonly used to monitor activity can seriously overestimate the effects of cosubstrate on cyclooxygenase velocity. The simulation results prompted re-examination of the effect of several cosubstrates (phenol, acetaminophen, N,N,N',N'-tetramethylphenylenediamine, and Trolox) on PGHS-1 cyclooxygenase velocity. Cyclooxygenase kinetics were examined at reduced temperature or elevated pH, where the oxygen electrode signal can be corrected to provide reliable oxygen consumption trajectories. The cosubstrates produced only a slight (10-60%) stimulation of the cyclooxygenase velocity. Peroxidase cosubstrates thus have a much smaller stimulatory effect on cyclooxygenase velocity than previously reported. This corrects a longstanding misperception of cosubstrate effects, provides more realistic kinetic constraints on PGHS mechanisms, and removes what was a major deficiency of branched-chain tyrosyl radical mechanisms.     

High frequency hearing loss correlated with mutations in the GJB2 gene

Genetic hearing impairment affects approximately 1/2000 live births. Mutations in one gene, GJB2, coding for connexin 26 cause 10%-20% of all genetic sensorineural hearing loss. Mutation analysis in the GJB2 gene and audiology were performed on 106 families presenting with at least one child with congenital hearing loss. The families were recruited from a hospital-based multidisciplinary clinic, which functions to investigate the aetiology of sensorineural hearing loss in children and which serves an ethnically diverse population. In 74 families (80 children), the aetiology was consistent with non-syndromic recessive hearing loss. Six different connexin 26 mutations, including one novel mutation, were identified. We show that GJB2 mutations cause a range of phenotypes from mild to profound hearing impairment and that loss of hearing in the high frequency range (4000-8000 Hz) is a characteristic feature in children with molecularly diagnosed connexin 26 hearing impairment. We also demonstrate that this type of audiology and high frequency hearing loss is found in a similar-sized group of deaf children in whom a mutation could only be found in one of the connexin 26 alleles, suggesting connexin 26 involvement in the aetiology of hearing loss in these cases. In our study of the M34T mutation, only compound heterozygotes exhibited hearing loss, suggesting autosomal recessive inheritance.     

Characterization of the heme environment in Arabidopsis thaliana fatty acid alpha-dioxygenase-1

Plant alpha-dioxygenases (PADOX) are hemoproteins in the myeloperoxidase family. We have used a variety of spectroscopic, mutagenic, and kinetic approaches to characterize the heme environment in Arabidopsis thaliana PADOX-1. Recombinant PADOX-1 purified to homogeneity contained 1 mol of heme bound tightly but noncovalently per protein monomer. Electronic absorbance, electron paramagnetic resonance, and magnetic circular dichroism spectra showed a high spin ferric heme that could be reduced to the ferrous state by dithionite. Cyanide bound relatively weakly in the ferric PADOX-1 heme vicinity (K(d) approximately 10 mm) but did not shift the heme to the low spin state. Cyanide was a very strong inhibitor of the fatty acid oxygenase activity (K(i) approximately 5 microm) and increased the K(m) value for oxygen but not that for fatty acid. Spectroscopic analyses indicated that carbon monoxide, azide, imidazole, and a variety of substituted imidazoles did not bind appreciably in the ferric PADOX-1 heme vicinity. Substitution of His-163 and His-389 with cysteine, glutamine, tyrosine, or methionine resulted in variable degrees of perturbation of the heme absorbance spectrum and oxygenase activity, consistent with His-389 serving as the proximal heme ligand and indicating that the heme has a functional role in catalysis. Overall, A. thaliana PADOX-1 resembles a b-type cytochrome, although with much more restricted access to the distal face of the heme than seen in most other myeloperoxidase family members, explaining the previously puzzling lack of peroxidase activity in the plant protein. PADOX-1 is unusual in that it has a high affinity, inhibitory cyanide-binding site distinct from the distal heme face and the fatty acid site.     

Tyrosine phosphorylation of myosin heavy chain during skeletal muscle differentiation: an integrated bioinformatics approach

Previously it has been shown that insulin-mediated tyrosine phosphorylation of myosin heavy chain is concomitant with enhanced association of C-terminal SRC kinase during skeletal muscle differentiation. We sought to identify putative site(s) for this phosphorylation event. A combined bioinformatics approach of motif prediction and evolutionary and structural analyses identified tyrosines163 and 1856 of the skeletal muscle heavy chain as the leading candidate for the sites of insulin-mediated tyrosine phosphorylation. Our work is suggestive that tyrosine phosphorylation of myosin heavy chain, whether in skeletal muscle or in platelets, is a significant event that may initiate cytoskeletal reorganization of muscle cells and platelets. Our studies provide a good starting point for further functional analysis of MHC phosphor-signalling events within different cells.     

A mechanistic study of self-inactivation of the peroxidase activity in prostaglandin H synthase-1

Prostaglandin H synthase (PGHS) is a self-activating and self-inactivating enzyme. Both the peroxidase and cyclooxygenase activities have a limited number of catalytic turnovers. Sequential stopped-flow measurements were used to analyze the kinetics of PGHS-1 peroxidase self-inactivation during reaction with several different hydroperoxides. The inactivation followed single exponential kinetics, with a first-order rate constant of 0.2-0.5 s-1 at 24 degrees C. This rate was independent of the peroxide species and concentration used, strongly suggesting that the self-inactivation process originates after formation of Compound I and probably with Intermediate II, which contains an oxyferryl heme and a tyrosyl radical. Kinetic scan and rapid scan experiments were used to monitor the heme changes during the inactivation process. The results from both experiments converged to a simple, linear, two-step mechanism in which Intermediate II is first converted in a faster step (0.5-2 s-1) to a new compound, Intermediate III, which undergoes a subsequent slower (0.01-0.05 s-1) transition to a terminal species. Rapid-quench and high pressure liquid chromatography analysis indicated that Intermediate III likely retains an intact heme group that is not covalently linked with the PGHS-1 protein.