The role of transition-state stabilization in enzyme catalysis, as proposed by Pauling, has been clearly demonstrated by extensive studies. In contrast, ground-state destabilization can also contribute to enzyme catalysis, but experimental evidence has been more limited. In recent years, high-resolution X-ray crystal structures of enzyme–substrate complexes have been obtained which show evidence for ground-state strain. We found that Y71F and F448H mutant tyrosine phenol-lyase (TPL) form complexes with 3-fluoro-l-tyrosine, a substrate, which shows a bending of the substrate aromatic ring about 20° out of plane, and we suggested that this was evidence for ground-state destabilization in TPL catalysis. Here, we have now evaluated quantitatively the role of ground-state destabilization in TPL catalysis. Phe-448 and Phe-449 are in close contact with the bound substrate side chain, and by mutating these residues to alanine and leucine, the contribution they play via ground-state destabilization was investigated. F448A, F448L and F449A TPL have activity for elimination of phenol from l-tyrosine reduced by a factor of 104, 103, and 104, respectively, but they have near-normal activity with the alternate substrates S-(o-nitrophenyl)-l-cysteine and S-ethyl-l-cysteine. F448A TPL forms quinonoid intermediates from l-tyrosine and S-ethyl-l-cysteine with rate constants similar to those of wild-type TPL. In addition, F448A TPL can form an aminoacrylate intermediate from S-ethyl-l-cysteine but not l-tyrosine, with a rate constant similar to that of wild-type TPL. Thus, the effect of the mutation is specifically on the elimination of phenol from l-tyrosine. We also examined the effect of hydrostatic pressure on the rates and equilibria of formation of the quinonoid intermediates from F448H and F448A TPL and 3-fluoro-l-tyrosine. Although the fastest phase shows only a small effect of pressure, the three slower phases have significant pressure dependences, suggesting that they may be associated with a conformational change. These results demonstrate that Phe-448 and Phe-449 contribute a total of about 108 to catalysis in TPL, about 50% of the estimated rate acceleration, by introducing ground-state destabilization into the l-tyrosine substrate.

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Background Bisphenol A (BPA) and Diethylhexyl Phthalate (DEHP) are common plastic‐derived environmental contaminants that act as endocrine disrupting chemicals (EDCs) and pose risks for expecting mothers and offspring. Research addressing the effect of a mixture of these EDCs is limited. Objective To assess the sex‐specific consequences of developmental exposure to individual as well as mixtures of environmentally relevant doses of BPA/DEHP in male and female Sprague‐Dawley rats. Methods From gestational day 6 till 21, dams were orally administered either saline (control), BPA (5mg/Kg BW/day), low‐dose (LD) DEHP (5mg/Kg BW/day), high‐dose (HD) DEHP (7.5 mg/Kg BW/day), a combination of BPA and LD DEHP (B+LD‐D), or a combination of BPA and HD DEHP (B+HD‐D). Gestational weights and number of abortions were tracked. Litter size and weights, number of live births and stillbirths were counted. Morphometric parameters at birth were measured. Body weighs, food and water intakes were monitored weekly from postnatal weeks 3–12. Male and female offspring were sacrificed at 16–24 weeks of age and organs were dissected and weighed. Results The abortion rate of dams exposed to HD DEHP and the mixtures, B+LD‐D and B+HD‐D were higher at 8, 14 and 23% respectively. Gestational index was reduced in these groups as well. Prenatal exposure to BPA or HD DEHP significantly decreased relative thymus weight in male but not female offspring at 16 weeks. Relative heart weight increased in B+HD‐D exposed male offspring compared to the other groups. No programming effects in relative organs weights were detected in female offspring. Conclusion The results indicate that a mixture of BPA and DEHP, even at low doses, induced a pronounced effect on pregnancy outcomes. Male offspring appear to be more susceptible to the programming effects of these EDCs or their mixture. This suggests a great need to reconsider the possible additive, antagonistic or synergistic effects of EDC mixtures to which pregnant individuals are commonly exposed to. Support or Funding Information Supported by UGA research foundation.