Paradigm for Improving the Catalytic Ability of Industrial Enzymes

Aug 30, 2007 - Paradigm for Improving the Catalytic Ability of Industrial Enzymes: Linkage Distortions of Carbohydrates in Complexes with Crystalline ...
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Chapter 15

Paradigm for Improving the Catalytic Ability of Industrial Enzymes: Linkage Distortions of Carbohydrates in Complexes with Crystalline Proteins Alfred D. French and Glenn P. Johnson Cotton Structure and Quality Research Unit, Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, L A 70124

Future innovations in applications of industrial enzymes to carbohydrates will require improved knowledge of the mode of action. One aspect of enzymatic hydrolysis of saccharides could be a twisting distortion of the bonds between adjacent monosaccharide residues in carbohydrate substrates. If such twists are important, then new enzymes could be engineered that would increase the distortion for faster reaction. One way to learn i f such distortion occurs is to survey existing crystalline carbohydrate-protein complexes. Unusual twists of linkages at the active site in an enzyme may result from catalysis. A related question is whether twisting of the linkage bonds increases the molecular potential energy. For the present work we have tracked the twisting in hundreds of protein-carbohydrate structures and used improved computer modeling of cellobiose to obtain energies. The largest apparent distortions were in similar molecules based on lactose.

U.S. government work. Published 2007 American Chemical Society.

Eggleston and Vercellotti; Industrial Application of Enzymes on Carbohydrate-Based Material ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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Introduction Emil Fischer's 1894 proposal (7) that enzymes and substrates form a "lock and key" relationship was an important step in the understanding of the mode of action of enzymes on their substrates, including carbohydrates. Further thinking resulted in Koshland's 1958 "induced fit" hypothesis (2), in which both enzyme and substrate were likely to change shapes as part of the overall reaction mechanism in question. In most situations, neither the substrates nor the enzymes were as rigid as originally envisioned. Work in 2001 by Bosshard (3) suggests that "induced fit" is not always entirely applicable for understanding interactions between enzymes and substrates that do not initially have complementary geometries. Instead, some combination of induced fit and conformational selection would be a better description of the interaction. At present, there is ample evidence of large-scale molecular motions that occur in some enzymes (4), and there is even a Database of Macromolecular Movements, http://www.molmovdb.org/ (5). However, detailed study of changes in shape of carbohydrate substrates has not been widely undertaken. In a 1966 landmark paper on the crystal structure of a complex of lysozyme and a pentasaccharide, Phillips wrote, "...the concerted influence of two amino acid residues, together with a contribution from the distortion to sugar residue D . . . is enough to explain the catalytic activity" (6). Phillips also stated that such ideas were not novel; that "...activation... by distortion has long been a favorite idea of enzymologists." Distortions could assist in enzymatic reactions by simply elevating the energy, thus putting the structure closer to the activation barrier. However, changed geometry might also make the substrate more vulnerable to attack, or changes in electronic structure resulting from geometric distortion might somehow facilitate the reaction. One elegant proposal suggested the importance of "orbital steering" that would align the parts of the molecules for reaction despite some energetic costs (7). At present, we are trying to understand whether types of distortion other than the monosaccharide ring geometry are identifiable and important. Knowledge of such details of the mechanism is essential to engineering enzyme proteins that could, for example, convert cellulose into liquid fiiels more rapidly and less expensively. Deliberate changes in the amino acid make-up of an enzyme could cause more distortion (or less!), in such a way that would possibly expedite the reaction. Distortion of molecules such as oligosaccharides could occur in several ways. In Phillips' paper (6), the distortion of the ring shape of residue D was obvious because it no longer had the chair form that is characteristic for such monosaccharides. Another type of distortion would be the internal twisting of a molecule about its inter-residue bonds. Less is known about what would constitute an abnormal, high-energy twist, and what, instead, is just another lowenergy molecular shape. This work considers a wide range of experimentally observed geometries of oligosaccharide substrates in crystalline

Eggleston and Vercellotti; Industrial Application of Enzymes on Carbohydrate-Based Material ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

209 complexes with proteins to see what is normal and what is unusual. We also emphasize calculated potential energies for the twisting through Ramachandran (8-9) mapping.

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 27, 2018 | https://pubs.acs.org Publication Date: August 30, 2007 | doi: 10.1021/bk-2007-0972.ch015

Distortion of

and \f/ as well as the numbering of carbon and ring and linkage oxygen atoms. We define cp and y/ (here