Substrate channeling, kinetics

Johnson and Fierke Hammes have presented detailed accounts of how rapid reaction techniques allow one to analyze enzymic catalysis in terms of pre-steady-state events, single-turnover kinetics, substrate channeling, internal equilibria, and kinetic partitioning. See Chemical Kinetics Stopped-Flow Techniques... 

Anderson, K.S. (1999) Fundamental mechanisms of substrate channeling, in Schramm, V. L. and Purich, D. L. (eds.), Methods in Enzymology 308, Enzyme kinetics and Mechanism, Part E, Academic Press, San Diego, pp. 111-145. 

It is crucial in performing TS analysis to know exactly which step of the reaction the experimental KIEs reflect. Using isotope-trapping experiments, it is possible to demonstrate whether formation of the Michaelis complex, E-S, is kinetically significant, and if necessary, to find conditions where it is not. However, internal steps can also complicate the interpretation of KIEs. These can include, but are not limited to (1) establishment of equilibria between different enzyme-bound intermediates, (2) isotopically insensitive steps, such as conformational changes in the enzyme or substrate, or (3) substrate channeling. 

Transient kinetic studies to understand substrate channeling 678... 

The primary sequence of the 0-subunit (397 residues) is also known, and extensive studies have been reported on the 3-dimensional structure of the 0202 multienzyme complex, in particular for the enzyme from Salmonella lyphimurium. The enzymes from Ecoli and S. lyphimurium are very similar the respective a-subunits both consist 268 amino acid residues and differ at 40 positions (15%), while the 0-monomers display only 3.5% difference in primary sequence (both consist of 397 residues, and only 14 of these are different). Crystallographic studies show that the active sites of the a- and 0-subunits are 25 A apart and connected by a tunnel, which presumably serves to carry the one metabolic intermediate (indole) from the active site of the a-subunit to the active site of the p-subunit. The kinetics of this substrate channeling have been studied by chemical quench-flow and stopped-flow methods- (I.P. Crawford J.Ito Proc. NatL Acad. ScL USA. 51 (1964) 390-397 B.P.Nichols CYanofeky Proc NatL Acad. Sci. USA. 76 (1979) 5244-5248 S-A. Ahmed et al. J. Biol. Chan. 260 (1985) 3716-3718 CCHyde et al. J. Biol. Chem. 263 (1988) 17857-17871 K.S. Anderson et al. J. Biol. Chem. 266 (1991) 8020-8033]... 

Wu, X., Gutfreund, H. Chock, P. B. (1992). Kinetic method for differentiating mechanisms for ligand exchange reactions application to test for substrate channeling in glycolysis. Biochemistry, 31,2123-8. 

---