Ketosteroid Isomerase Catalysis

Understanding the mechanisms by which enzymes catalyze chemical reactions with enormous rate enhancements and exquisite specificity is central to biology. And knowledge of the chemical and physical basis of catalysis provides fundamental information that may facilitate the design of novel, artificial enzymes and aid the design of enzyme inhibitors that may act as drugs.

There have been astounding advances in the understanding of enzyme mechanism over the past decades. Nevertheless, fundamental questions remain, and indeed, much of the previous work has helped to bring these critical questions into focus.

Enzymes cannot be described as simply a collection of catalytic amino acids and cofactors. Rather, these groups come together with a precise active site arrangement in a unique, idiosyncratic environment created by the protein scaffold. It is the large number of interactions and this ‘context’ that distinguish enzymes from simple, small molecule catalysts and that must be explored to achieve the next level of understanding of enzymatic catalysis.

The bacterial enzyme ketosteroid isomerase (KSI) provides a remarkably tractable system for in-depth dissection of the mechanisms of catalysis used by enzymes: It catalyzes a simple, well-characterized chemical transformation; it has an established kinetic and thermodynamic reaction framework; it has available transition state analogs to allow structural and spectroscopic probes of active site interactions; it has known, high resolution x-ray crystal structures and is amenable to rapid determination of X-ray structures of new variants and complexes; it catalyzes equilibration of a single substrate/single product reaction, allowing enzyme/substrate complexes to be observed by crystallography and other structural techniques; it is amenable to a panoply of biophysical probes, including NMR and vibrational spectroscopy; and it can be generated semi-synthetically to allow the incorporation of series of unnatural amino acids to systematically and rationally test specific mechanistic proposals.

Some leading papers from the lab in the areas of enzyme mechanism and ketosteroid isomerase are:

Kraut, D.A., Carroll, K.S. and Herschlag, D. (2003) Annu. Rev. Biochem. 72, 517-571. “Challenges in Enzyme Mechanism and Energetics.” (Medline) (PDF File)

Kraut, D.A., Sigala, P.A., Pybus, B., Liu, C.W., Ringe, D., Petsko, G.A. and Herschlag, D. (2006) PloS Biology 4 e99 “Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole.” (Medline) (PDF File)

Sigala, P.A., Fafarman, A.T., Bogard, P.E., Boxer, S.G. and Herschlag, D. (2007) J. Am. Chem. Soc. 129, 12104-12105. “Do Ligand Binding and Solvent Exclusion Alter the Electrostatic Character within the Oxyanion Hole of an Enzymatic Active Site?” (Medline) (PDF File)
See also: Faculty of 1000 Biology, 16 Oct. 2007 http://www.f1000biology.com/article/id/1091226/evaluation

Sigala, P., Kraut, D., Caaveiro, J., Pybus, B., Ruben, E., Ringe, D., Petsko, G., Herschlag, D., (2008) J. Am. Chem. Soc. 130, 13696-13708. “Testing Geometrical Discrimination within an Enzyme Active Site: Constrained Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole.”(Medline) (PDF File)
See also: Nature 2008, 456, 45-47. http://www.nature.com/nature/journal/v456/n7218/full/456045a.html

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