Enzymes substrate trapping experiments

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

G. Strategies for Trapping Crystalline Enzyme—Substrate Complexes 1. Preliminary Experiments... 

Conclusively establishing the role of potential intermediates in a biosynthetic pathway is a difficult aspect of biosynthesis. Typically, intermediates accumulate because subsequent enzymatic reactions are slow. Organisms also produce shunt metabolites that are off the main pathway and may not be further metabolized these will also accumulate. Isolation of an intermediate does not, therefore, establish intermediacy. Trapping experiments are sometimes used to overcome these problems. In the pathway A B C, where A is a known precursor of C, labeled A and non-labeled B are fed at the same time. The latter is metabolized to C and labeled B is produced from A Bis then temporarily available for isolation. An alternative approach for microbial metabolites is to mutate the organism or add specific enzyme inhibitors. This may allow intermediates to accumulate. Incorporation of a labeled, potential intermediate into a product does not prove that the intermediate lies on the main biosynthetic pathway. It may simply serve as a substrate for the enzymes involved. Only when each of the enzymes in a pathway has been isolated and characterized, and the substrate specificity determined, can the intermediates in a biosynthetic route be characterized. 

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 method of isotope trapping allows one to detemrine the stickiness of all substrates but the last to combine in an ordered mechanism and of all substrates in a random mechanism (Rose et al, 1974 Rose 1995). This method was developed initially by Rose for the yeast hexokinase reaction, and it is essentially a single turnover experiment in which one detemtines analytically the proportion of an enzyme-substrate complex that reacts to give products, as opposed to dissociating. 

Furthermore, it is difficult to incorporate the EPSP ketal as an intermediate in a chemically plausible reaction mechanism for converting S3P and PEP into EPSP and P . In the experiments described above using solution NMR and C-2 PEP to observe the tetrahedral intermediate on the enzyme at internal equilibrium with substrates and products, the EPSP ketal was formed over longer times as a dead-end breakdown product of tetrahedral ketal phosphate intermediate. " The formation of EPSP ketal most likely occurs through trapping of a protonated enol form of PEP using the 4-OH group of S3P as a nucleophile. It is believed that this side product is also observed in a solid-state NMR experiment."" ... 

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