Type of Document	Dissertation
Author	Laxmikanthan, Gurunathan
Author's Email Address	laxmikanthan@chem.fsu.edu
URN	etd-09252006-192617
Title	An Insight into the Structure, Function and Activation Cascades of Classical Human Kallikreins
Degree	Doctor of Philosophy
Department	Chemistry and Biochemistry, Department of
Advisory Committee	Advisor Name Title Michael Blaber Committee Chair Albert Stiegman Committee Member Hong Li Committee Member Thomas C.S. Keller III Committee Member
Keywords	Induced Fit Activation X-Ray Structure Human Kallikrein 1 Proform Of Kallikreins Cascade
Date of Defense	2006-09-20
Availability	unrestricted
Abstract This work discusses the structure, function, activation and inhibitor screening of human kallikrein 1. Human kallikrein 1 is the first member of the kallikrein family and is involved in blood pressure regulation. hK1 exhibits unique dual-substrate specificity, and is able to hydrolyze low molecular weight kininogen between both Arg-Ser and Met-Lys sequences. In the chapter 2, the x-ray crystal structure of mature active recombinant hK1 is presented. The active site exhibits structural features intermediate between that of apo and pro forms of known kallikrein structures. The S2 to S2’ pockets demonstrate a variety of conformational changes in comparison to the porcine homolog of K1 in complex with peptide inhibitors, including the displacement of an extensive solvent network. These results indicate that the binding of a peptide substrate contributes to a structural rearrangement of the active-site Ser 195 resulting in a catalytically competent juxtaposition with the active-site His 57. The activation of proforms of kallikreins has not been well characterized but it has been postulated that some kallikreins can autoactivate and some might be cross-activated by other members of the family. In the chapter 4, in order to help elucidate the details of activation of the human kallikreins, including the potential for activation cascades, we have constructed kallikrein pro-peptides fused to an appropriate soluble carrier protein. The pro-peptide fusion proteins corresponding to pro-hK1, pro-hK2 and pro-hK3 (i.e. the “classical” kallikreins) have been characterized with regard to their proteolytic processing by active recombinant hK1, hK2 and hK3. The results are in agreement with prior reports that mature hK2 can activate pro-hK2 (i.e. hK2 can auto-activate) as well as pro-hK3 (illustrating kallikrein cross-activation). Additionally, the results provide new information showing that hK2 can activate pro-hK1, and also demonstrate the potential for hK3 to exhibit weak trypsin-like dual-substrate specificity and activate the pro-forms of hK1, hK2 and hK3. The chapter 5 discusses about the virtual screening process performed by Autodock against the National Cancer Institute’s Diversity set database of two thousand compounds. The screening has resulted in the generation of potential hits which can be used as leads for development of oral medications.
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