Substrate trapping

In 2002, Grubbs and co-workers reported the first CM reactions of allyl phosphines.In an initial reaction, subjecting allyl diphenylphosphine to catalyst 5 (5 mol%) failed to produce any of the desired cross-product. However, by protecting the phosphine as its borane complex, CM reactions could be achieved in good yield with high E-selectivity (Equation (5)). Notably, catalyst 5 failed to dimerize borane-protected vinyl diphenylphosphine. This result was attributed to substrate trapping of the catalyst as an unreactive Fischer carbene, a situation analogous to that observed in the CM reactions of alkyl vinyl ethers. 

Pfeifer, Wely, and Wippermann1671 examined surface portions of a lysozyme protein molecule. A fractal dimension of D = 2.17 was determined and related to the rate of substrate trapping at the enzyme active site. The root mean square substrate displacement via diffusion at the surface increases as time (t)1AD whereas surface capture of substrates is greater when compared with a smooth (D = 2) surface. From their results, the authors suggested that the observed value of D = 2.17 corresponds to an optimum overall capture rate. 

SUBSTRATE TRAPPING IN CYSTEINE TO SERINE MUTATED PTPases... 

The cysteine residue in the catalytic loop (Cysl2 in LMPTP, Cys215 in PTPIB) is the essential nucleophile for PTPase activity. Experiments show that cysteine to serine mutants are completely inactive but can still bind substrate molecules. The ability to bind substrates without hydrolyzing them is called substrate trapping and has been exploited when searching for native PTPase... 

Xie L, Zhang YL, Zhang ZY (2002) Desi and characterization of an improved protein tyrosine phosphatase substrate-trapping mutant. Biochemistry 41 4032-4039... 

Flint AJ, Tiganis T, Barford D et al (1997) Development of substrate-trapping mutants to identify physiological substrates of protein tyrosine phosphatases. Proc Natl Acad Sci USA94 1680-1685... 

Blanchetot C, Chagnon M, Dube N et al (2005) Substrate-trapping techniques in the identification of cellular PTP targets. Methods 35 44-53... 

Agazie YM, Hayman MJ (2003) Development of an efficient substrate-trapping mutant of Src homology phosphotyrosine phosphatase 2 and identification of the epidermal growth factor receptor, Gabl, and three other proteins as target substrates. J Biol Chem 278 13952-13958... 

Measure the rates of substrate binding and dissociation by stopped-flow methods or by substrate trapping methods. This is an optional step in that although information on binding rates can be useful, it is not essential for the design of subsequent experiments. 

It should also be noted that a multisubstrate enzyme also allows the application of substrate trapping methods to estimate the rates of dissociation of the first substrate to bind to the enzyme. These methods will be described in Section IV,F,2. 

In the example involving the binding of S3P and glyphosate, it was relatively easy to resolve the two-step reaction kinetics because of the separate concentration dependence for each step the first step depended on S3P and the second on glyphosate. Moreover, substrate trapping studies established that S3P dissociates at a very fast rate (i), justifying the rapid equilibrium assumption. With the... 

For a sequential bisubstrate enzyme, the rate of dissociation of the hrst substrate can be estimated by substrate trapping methods. The rationale for this experimental approach is shown in Scheme XXII. The enzyme is first preincubated with radiolabeled substrate A and is then mixed with an excess of unlabeled substrate A and substrate B to initiate the reaction. The recovery of radio-labeled product is a function of the kinetic partitioning of the enzyme-bound substrate between dissociation to yield free S and forward reaction with substrate B to yield product P. 

Results listed in Table 20 show that the values of e.e. are low in the presence of excess substrate, which indicates that the reaction of BSA-S-NAH with the free substrate does not exert stereoselectivity. This is in contrast to the fact described above that the reaction between a substrate trapped on BSA and a free reactant (NaBH or... 

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