Biochemistry: The Chemical Reactions of Living Cells, Vol. 2 - D. Metzler 1980

Enzymes: Cellular Protein Catalysts
Enzyme Specificity
Complementarity of Substrate and Enzyme Surfaces

The striking Specificity of Enzyme Action led to the formulation of the lock-and-key theory, according to which a reaction requires a precise structural match between the substrate and the enzyme's Active Site. Experiments have convincingly demonstrated the validity of this concept, though the theory itself has undergone substantial revision. It is now understood that if the enzyme is the "lock" and the substrate is the "key," inserting the key into the lock often induces Conformational Changes in the protein molecule. Numerous studies have shown that the enzyme "Molds" itself around the substrate, ensuring a more precise fit between the interacting structures. This is supported by data on changes in circular dichroism spectra, UV absorption spectra, and sedimentation constants, as well as by X-ray crystallographic analyses of enzyme-inhibitor complexes. As we have seen previously (Ch. 4, Sec. D, I), METABOLISM/2.html">THE CONCEPT OF "induced fit" also proves highly productive when discussing subunit interactions.

What is The Structure of active sites? Crystallographic studies allow us to directly "visualize" the architecture of a growing number of them. However, X-ray crystallography generally does not provide a clear picture of the conformational changes responsible for the induced fit. Furthermore, high-resolution crystallographic data are available for only a relatively small number of Enzymes. Therefore, to elucidate active site structure, enzymologists continue to rely heavily on traditional chemical "mapping" Methods. These involve measuring binding constants for inhibitors with systematically altered structures, and investigating how structural modifications of substrates affect binding and reaction rates. A prime example of this approach is the work of Meister and coworkers on sheep Brain Glutamine Synthetase. The substrates for this enzyme include both D- and L-glutamic acids and a-aminoadipic acid. At the same time, out of ten monomethyl derivatives of D- and L-glutamic acids, only three can serve as substrates for glutamine synthetase. Assuming that substrates bind in a fully extended conformation, all hydrogen atoms whose replacement does not abolish activity lie on one side of the molecular backbone (behind the plane of the page in the following two diagrams):

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When the hydrogen atoms circled in the diagrams are replaced, the compound retains its ability to act as a substrate for glutamine synthetase. This indicates that the face of the molecule presented to the enzyme is the one facing the reader in the diagrams. Building on this hypothesis, Gass and Meister [43] synthesized a dicarboxycyclohexane analog of L-glutamate (L-cis-1-amino-1,3-dicarboxycyclohexane):

In this analog, the cyclohexane ring bridges the positions previously occupied by the two replaceable hydrogen atoms. The synthesized compound proved to be a good substrate for glutamine synthetase, thereby corroborating the authors' hypothesis. Moreover, these data provided specific insights into the geometry of the enzyme's binding site.