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.