α-Lytic protease (α-LP), an extracellular bacterial serine protease in the chymotrypsin family, serves as an excellent model for studying the mechanisms of proteolytic catalysis and protein folding/stability. α-LP forms crystals that diffract to ~ 0.80 Å resolution. At sub-angstrom resolution, the positions of all atoms (including hydrogen atoms) can be determined accurately. During my graduate work, I solved the structure of α-LP at 0.83Å resolution (1SSX.pdb). Surprisingly, this structure revealed the distortion of a core residue, Phe228, suggesting a mechanism by which α-LP achieves its extreme kinetic stability (“Kinetic Stability of Bacterial Proteases” research summary).

My current research focuses on the proteolytic mechanism of α-LP. By analyzing ultra-high resolution crystal structures of α-LP transition state analogs, I am addressing these questions:

* Is there a low-barrier hydrogen bond between His57 and Asp102 in the transition state?
* As one lengthens serine protease peptide substrates from one to four amino acids (filling the S1 – S4 binding pockets, respectively), the catalytic efficiency of the proteolytic reaction increases by 104. Curiously, this increase in catalytic efficiency (kcat / Km) is accounted for almost entirely by an increase in the kcat. How is binding energy being translated directly into stabilization of the transition state?

My current work is an extension of my graduate work in David Agard's lab (Ph.D. awarded September 2005).

Fuhrmann, C.N., Kelch, B.A., Ota, N., Agard, D.A. "The 0.83Å resolution crystal structure of α-lytic protease reveals the detailed structure of the active site and identifies a source of conformational strain," J. Mol Biol. (2004);338(5):999-1013. (html or pdf)

Fuhrmann, C.N., Ota, N., Rader, S.D., Agard, D.A. (2004) " α-Lytic protease," in Handbook of Proteolytic Enzymes, 2nd edition (Barrett, A.J., Rawlings, N.D., Woessner, J.F., eds.), Academic Press, Cambridge.

Cheng, A.C., Chen, W.W., Fuhrmann, C.N., Frankel, A.D. (2003) "Modeled databases of amino acid-base and amino acid-base pair hydrogen-bonding interactions," J. Mol. Biol., 327, 781-96.

PDB Codes

1SSX.pdb -- 0.83Å resolution crystal structure of α-lytic protease at pH 8

Research Presentations

“Ultra-high resolution x-ray crystallography of α-lytic protease: Exploring the static and dynamic properties of enzyme catalysis at 0.86Å resolution.” Fuhrmann, C.N., Waddling, C.A., Shipley, K.M., Agard, D.A. Invited Seminar Speaker, Medical College of Wisconsin (August 2002).

"A new view: Sub-angstrom resolution studies of α-lytic protease." Fuhrmann, C.N., Waddling, C.A., Shipley, K.M., Agard, D.A. Oral presentation, West Coast Protein Crystallography Workshop (March 2001).