Enzymes 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. 

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]... 

SECM can be applied to imaging and kinetic studies of biological systems. Ground work on enzymes, ion channels, and cellular system has been reported and demonstrates SECM capabilities. To use SECM in diagnostic assays, nucleic acid analysis, biosensor, bioremediation or other biotechnological processes, SECM needs to be applied quantitatively to different biological systems. There is also a need for the development of accessible kinetic theories, the development of controlled substrate methodologies, and the fabrication of smaller well-characterized UMEs that would increase the lateral resolution of SECM. 

For in vitro assays, it is extremely difficult to determine the relevant intracellular concentrations of substrates due to compartmentalization of particular reactions, competing reactions that produce and consume common substrates, and metabolic channeling (Albe et al, 1990 Srere, 1987). Consequently, in vitro kinetic measurements may not be very useful for assessing in situ or in vivo reaction rates. However, provided assays conditions are carefully controlled, kinetic parameters may be very useful for characterizing enzyme isoforms and enzymes from different metabolic pools, different organisms, or from organisms collected from different environments. 

Such a macroscopic conceptualization of concentration breaks down for microheterogeneous enzyme systems in organized states, wherein the kinetically competent value of concentration for the intermediary metabolites takes on a local, anisotropic character that is part-and-parcel of the organization itself. For structured systems that tightly channel the intermediates on a one-by-one basis, the notion of concentration may not even apply, kinetically speaking, except for the initial substrate and the final product. Commensurate with this picture, the measured concentrations of many metabolites are found to be of the same order of magnitude as that of their cognate enzymes (Srere, 1987). 

Three features of the reaction kinetics are essential to ensure that indole is channeled efficiently (1) the reaction of serine at the f3 site modulates the formation of indole at the a site such that indole is not produced until serine has reacted to form E AA (2) the rate of reaction of indole and E AA is fast and largely irreversible and (3) the rate of indole diffusion from the a site to the f3 site is very fast (>1000 s ). ° This mechanism accounts for the fact that indole does not accumulate during a single turnover of conversion of IGP into tryptophan (the af3 reaction). This model makes several predictions, which have been tested by kinetic and structural analysis of mutants and alternate substrates ° using single enzyme turnover experiments. 

The membrane-bound ATP(synth)ase affinity (Michaelis constant K ) for its substrates was found quite variable for ADP, from less than T yM (1) to almost 200 yM (2). In fact, this was predictable (3), inasmuch as the chemiosmotic mechanism of phosphorylation makes that, as soon as ADP is added, the proton channels open, the proton gradient A]5f + lowers, and consequently the catalytic constant and/or the enzyme number, because of their AvL+-dependent activation (4), decrease. That is, is not constant in the kinetic determination of K. ... 

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