Chemical quench experiments

A series of rapid chemical quench experiments under single enzyme turnover conditions using radiolabeled S3P or PEP revealed that the tetrahedral ketal phosphate enzyme intermediate was formed as a new peak upon HPLC analysis with detection of the radiolabel. The time course revealed that the formation of the tetrahedral intermediate species paralleled the disappearance of PEP substrate and formation of the EPSP product thus establishing that it was a kinetically competent species. Isolation of the tetrahedral ketal phosphate intermediate using C-2 PEP and S3P as substrates coupled with rapid chemical quench was carried out in conjunction with H-, C-, and P- NMR to provide a definitive structure proof Thus with these studies we have satisfied the criteria for a true reaction intermediate in terms of a chemically plausible mechanism, structure proof, and kinetic competence. Additional studies support the mechanism for EPSP synthase described (Scheme 4, pathway a) including observation of the intermediate bound to the enzyme at internal equilibrium using solution NMR and C-2 PEP as well as using rapid freeze-quench/solid-state NMR studies. ... 

The kinetics of the formation and decay of the observed intermediates through the formation of Q show no concentration dependence on either O2 or methane [15, 26] (O formation is presumably dependent on O2 concentration, but this has not been directly confirmed). In contrast, Q decay is linearly dependent on methane concentration indicating that this is the step where substrates react with the enzyme. Other substrates also accelerate Q decay, and the second order rate constant for the process depends on the specific substrate used. When nitrobenzene is used as a substrate, the decay of Q leads to the formation of another chromophoric intermediate, which we term compound T (T) for the terminal complex [15]. Chemical quench experiments showed that T is the nitrophenol (product) complex of... 

Pyrene Solution Preparation. Pyrene was purchased fi om Aldrich Chemical Company and used directly. A phosphate buffer, pH 6 upon dilution was used (6). For the fluorescence quenching experiments an aqueous pyrene stock solution was prepared fi-om a 2.05 x 10" solution of pyrene in methanol 1.5 ml of the methanolic pyrene solution was mixed with 498.5 ml of pH 6 buffer water to give a concentration of 6.15 X 10" M aqueous pyrene. The pyrene fluorescence intensity tended to be more stable if the solution were allowed to mix 1 hour before use. 

It was initially assumed that the degradation of the phenacyl aryl ether bond occurred through the u-tt triplet excited state [14-18]. However, quenching experiments and time-resolved chemically induced dynamic nuclear polarization (CIDNP) spectra have indicated that there is significant photodegradation through the singlet excited state [23-27]. 

Chemical evidence for the radical cation, 7, can be obtained from quenching experiments. A known electron transfer quencher,... 

Perhaps the most difficult aspect of learning transient-state kinetic methods is that it is not possible to lay down a prescribed set of experiments to be performed in a given sequence to solve any mechanism. Rather, the sequence of experiments will be dictated by the details of the enzyme pathway, the relative rates of sequential steps, and the availability of signals for measurement of rates of reaction. The latter constraint applies mainly to stopped-flow methods, and less so for chemical-quench-flow methods provided that radiolabeled substrates can be synthesized. Therefore, 1 will describe the kinetic methods used to establish an enzyme reaction mechanism with emphasis on the direct measurement of the chemical reactions by rapid quenching methods. Stopped-flow methods are useful in instances in which optical signals provide an easy means to measure the rates of individual steps of the reaction. 

The most serious limitation of stopped-flow methods is that one does not always have an optical signal for the reaction of interest and the optical signals cannot be interpreted rigorously if the extinction coefficients of intermediates or products are not known. For example, an enzyme intermediate may have an unknown extinction coefficient, and without an absolute measurement of concentration of the intermediate, one cannot obtain a unique solution to its rate of formation and decay (see below). For these reasons, a direct measurement of the conversion of substrate to product is required. Chemical-quench-flow methods allow such direct measurement of the chemistry of enzyme-catalyzed reactions and can be performed for nearly any reaction. One must recognize that these experiments are based on examining the enzyme as a stoichiometric reactant such that the concentration of enzyme required will depend upon the kinetics of the reaction and the sensitivity of the methods for detection of intermediates or products. Nonetheless, quench-flow experiments can be performed using as little as 20 fd of solution and a complete enzyme pathway can be solved using only... 

Pulse-chase/pulse-quench experiments with KF ° indicated accumulation of the nucleotide bound enzyme species, which would not be possible if the forward reaction was much faster than the rate of conformational closing. To explain this observation, the authors proposed the presence of a kinetic road block - a slow step after the phosphodiester bond formation. However, the results of the pulse-chase/ pulse-quench experiments can also be explained by designating chemistry as the slow step, meaning that the chemical step itself plays the role of the road block. The conclusion that chemistry is a fast step in the KF reaction pathway was made based on the observation of a small thio-efifect magnitude,which, as elaborated in the following section, should not be used as a solid evidence of the chemical step being nonrate limiting. 

K. S. Anderson, Deteotion and Charaoterization of Enzyme Intermediates Utility of Rapid Chemical Quench Methodology and Single Enzyme Turnover Experiments. In Kinetic Analysis of Macromolecules Practical Approach Series] K. A. Johnson, Ed. Oxford University Press, Ino. New York, 2003 Vol. 267, pp 19-44. 

However, the chemical results alone indicate that migration of silyl groups from O to N is very rapid in hydroxylamine anions. The results of quenching experiments at -78°C show that the half-life for rearrangement cannot be more than a few minutes at this temperature. 

Hynne, F. Sprensen, P. Quenching of chemical oscillations. J. Phys. Chem. 1987, 91, 6573-6575 Sprensen, R Hynne, R Amplitudes and phases of small-amplitude Belousov-Zhabotinskii oscillations derived from quenching experiments. J. Phys. Chem. 1989, 93, 5467-5474. 

The rate constant for total deactivation of singlet oxygen by Vaska s Complex + kq) was determined by IO2 luminescence quenching and is listed in Table 1. In order to obtain the rate constant of chemical quenching (k ), competition experiments were carried out with 9,10-dimethylanthracene (DMA, same conditions as above). DMA is known to quench IO2 only chemically. " Loss of 1 and DMA were monitored spectrophoto-metrically and the results fitted to the equation of Higgins et al A second independent determination of was obtained by measurements of direct disappearance of 1 compared with a tetramethylethylene (TME) reference, which quenches singlet oxygen only by a chemical mechanism. Values of / r, and by difference, of kq, are listed in Table 1,... 

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