Protein transition states

Houk KN, Leach AG, Kim SP, Zhang X. Binding affinities of host-guest, protein-ligand, and protein-transition-state complexes. Angew Chem Int Ed 2003 42 4872-4897. 

A Tropsha, J Hermans. Application of free energy simulations to the binding of a transition-state-analogue inhibitor to HIV protease. Protein Eng 51 29-34, 1992. 

A Li, V Daggett. Characterization of the transition state of protein unfolding by use of molecular dynamics Chymotrypsm inhibitor 2. Proc Natl Acad Sci USA 91 10430-10434, 1994. 

Recently Alan Fersht, Cambridge University, has developed a protein engineering procedure for such studies. The technique is based on investigation of the effects on the energetics of folding of single-site mutations in a protein of known structure. For example, if minimal mutations such as Ala to Gly in the solvent-exposed face of an a helix, destabilize both an intermediate state and the native state, as well as the transition state between them, it is likely that the helix is already fully formed in the intermediate state. If on the other hand the mutations destabilize the native state but do not affect the energy of the intermediate or transition states at all, it is likely that the helix is not formed until after the transition state. 

Fersht, A.R. Characterizing transition states in protein folding an essential step in the puzzle. Curr. Opin. Struct. Biol 5 79-84, 1994. 

In this chapter we shall illustrate some fundamental aspects of enzyme catalysis using as an example the serine proteinases, a group of enzymes that hydrolyze peptide bonds in proteins. We also examine how the transition state is stabilized in this particular case. 

The active site of subtilisin is outside the carboxy ends of the central p strands analogous to the position of the binding sites in other a/p proteins as discussed in Chapter 4. Details of this active site are surprisingly similar to those of chymotrypsin, in spite of the completely different folds of the two enzymes (Figures 11.14 and 11.9). A catalytic triad is present that comprises residues Asp 32, His 64 and the reactive Ser 221. The negatively charged oxygen atom of the tetrahedral transition state binds in an oxyanion hole,... 

Transition-state stabilization in subtilisin is dissected by protein engineering... 

Rittinger, K., et al. Structure at 1.65 A of RhoA and its GTPase-activating protein in complex with a transition-state analogue. Nature 389 758-762, 1997. 

A structural anomaly in subtilisin has functional consequences Transition-state stabilization in subtilisin is dissected by protein engineering Catalysis occurs without a catalytic triad Substrate molecules provide catalytic groups in substrate-assisted catalysis Conclusion Selected readings... 

Lolis, E., and Petsko, G., 1990. Transition-state analogues in protein crystallography Probes of the structural source of enzyme catalysis. Annual Review of Biochemistry 59 597—630. 

Wolfenden, R., and Kati, W. M., 1991. Testing the limits of protein-ligand binding discrimination widi transition-state analogue inhibitors. Accounts of Chemical Research 24 209-215. 

While recent attention has been largely on proteins, it should be borne in mind that membrane fusion ultimately involves the merger of phospholipid bilayers. However, little is known about the specific membrane lipid requirements. When membranes fuse, energetically unfavorable transition states are generated that may require specific lipids and lipid domains for stabilization. Although there is some evidence for a specific influence of lipids on exocytosis, it is still unclear whether specific lipid metabolites are needed or even generated at the site of membrane merger. 

FIGURE 6.6. The type of model compounds that were used to estimate the electrostatic stabilization in lysozyme (the only hydrogen atom shown, is the one bonded to the oxygen). Such molecules do not show a large rate acceleration due to electrostatic stabilization of the positively charged carbonium transition state. However, the reaction occurs in solution and not in a protein-active site, and the dielectric effect is expected to be very different in the two cases. 

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