Rapid chemical quench

Is the paramagnetic adduct between CO and Cluster A a kinetically intermediate in acetyl-CoA synthesis Questions have been raised about whether this adduct is a catalytic intermediate in the pathway of acetyl-CoA synthesis 187, 188) (as shown in Fig. 13), or is formed in a side reaction that is not on the main catalytic pathway for acetyl-CoA synthesis 189). A variety of biochemical studies have provided strong support for the intermediacy of the [Ni-X-Fe4S4l-CO species as the precursor of the carbonyl group of acetyl-CoA during acetyl-CoA synthesis 133, 183, 185, 190). These studies have included rapid ffeeze-quench EPR, stopped flow, rapid chemical quench, and isotope exchange. 

More direct approach to the problem is based on measuring rapid presteady state kinetics with the use rapid chemical quench and stop-flow techniques (Johnson, 1995 Fierke and Hammes, 1995). These techniques allow monitoring individual rates of binding, conversion and dissociation of substrate. The most effective variant of such an approach is based on using a single turn over kinetics in which enzyme is taken in excess over radiolabeled substrate. 

The two prominent transient-kinetic mixing methods are stopped flow and rapid chemical quench flow. In the stopped flow, the... 

Houston et al,12 reported the capability to measure pre-steady state kinetics using rapid chemical quench-flow methods coupled with MALDI-TOF-MS. However, they reported difficulties in obtaining accurate pre-steady state... 

Pre-steady-state kinetics using discontinuous assays (rapid chemical quench) 355... 

Under pre-steady-state conditions, the enzyme in the reaction is used in stoichiometric amounts, meaning that one of the substrates has a concentration smaller than or comparable to the enzyme concentration. In single-turnover DNA polymerase assays of dNTP incorporation, the enzyme concentration is in excess of the DNA substrate concentration. These conditions allow us to follow the enzyme through one complete catalytic cycle, thus eliminating complications from multiple turnovers. Nucleotide incorporation is a relatively fast process occurring on a millisecond timescale, and rapid chemical quench is the specialized instrument generally... 

Figure 5 Typical rapid chemical quench assay of single-nucleotide incorporation by Pol j3. (a) Single-turnover time course of DNA product formation fitted to a single exponential equation [DNAn+i] =A (b) Plot of rate versus...

Figure 10 Superimposition of rapid chemical quench (open circles O) and stopped-flow fluorescence (blue) assays. In (a) tryptophan emission was detected, and in (b) the fluorescence change from 2-AP was monitored. Insets show the dNTP binding-induced conformational change In the presence of dideoxy-terminated DNA substrate. Adapted with permission from A. K. Showalter B. J. Lamarche M. Bakhtina M. I. Su K. H. Tang M. D. Tsai, Chem. Rev. 2006, 106, 340-360. Copyright 2006 American Chemical Society.

Figure 13 Comparison of Pol /3 catalyzed single-nucleotide incorporation at 10% and 35% glycerol, pH 8.3. (a) 2-AP fluorescence stopped-flow assays show that both phases of the fluorescence change are slowed down at increased glycerol concentration, (b) In contrast, rapid chemical quench assays demonstrate that the rate of nucleotide incorporation remains unaffected by the altered glycerol concentration. Adapted with permission from M. Bakhtina M. P. Roettger S. Kumar ...

Coupling of rapid chemical quench or freeze-quench methods with ERR, MS, or NMR has been a very useful strategy for detecting reaction intermediates that are paramagnetic such as transition metal complexes in metalloenzymes and radical species " " or to determine the chemical structure of transient reaction intermediates or reaction products." ... 

Rapid chemical quench has been enormously powerful and helped to open the field of transient enzyme catalysis, as described above however, an alternative strategy is required for the detection and characterization of labile intermediates that avoids the need for chemical quenching. The coupling of rapid mixing techniques with online MS detection not only offers an option for the detection of labile intermediates but also has many general applications in the study of enzyme catalysis to more precisely define the key chemical events occurring at the active site. 

This, in fact, was the case for detection of a hemiketal phosphate intermediate formed in the KD08P synthase reaction pathway described in Section 8.18.5. Other potential limitations of rapid chemical quench methodology include the need for radiolabeled substrates and the need for subsequent analytical techniques, for example, NMR, to elucidate structural information on enzyme intermediates. An earlier article focusing on New Concepts in Bioorganic Chemistry has highlighted the potential of using high-resolution MS coupled with electrospray ionization (ESI) to examine rapid enzyme reactions. ... 

In this section, we discuss the (1) application of the concepts of rapid chemical quench studies to detect enzyme intermediates, (2) biological or medical interest of studying these enzymes, and (3) the underlying kinetic concepts behind these studies. The examples chosen all perform unique, novel chemistries, and have proven to be exciting pharmaceutical targets both for antibiotics and anticancer drugs as well as herbicides. 

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. ... 

In summary, through the use of rapid chemical quench techniques, multiple studies demonstrated the formation of a single tetrahedral intermediate in the reaction pathway of EPSP synthase (Scheme 4, pathway a) which is formed by an attack of the 5-OH group of shikimate-3-phosphate on C-2 of PEP. A complete kinetic and thermodynamic description of this enzyme reaction pathway could be demonstrated, including the isolation and structural elucidation of a tetrahedral enzyme intermediate as originally proposed by Sprinson. This work established the catalytic mechanism and definitively showed that no covalent enzyme—PEP adduct is formed on the reaction pathway. Subsequent work using rapid mixing pulsed-flow ESI—MS studies and solution phase NMR " provides additional support for the catalytic pathway in Scheme 4, pathway a. 

The proposed cyclic ketal phosphate intermediate 2 in Scheme 6 has very similar functionality to the tetrahedral enzyme intermediates isolated and characterized in the EPSP synthase and UDP-GlcNAc enol-pyruvoyl transferase reactions. Although the tetrahedral ketal phosphate intermediate for EPSP synthase is quite labile at neutral or acidic pH, it is surprisingly stable at basic pH>12 (q/2=48h). Based upon these results, we might predict that intermediate 2 should be detectable by rapid chemical quench techniques if isolated under basic conditions even if it was formed only transiently at the enzyme active site. Although the mechanistic data described above suggest the catalytic pathway outlined in Scheme 6, there was no direct information in support of either intermediate. 

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. 

The exchange of oxygen between iodate ions and water has been investigated by a rapid chemical-quenching technique.Over the pH range 2.1—12.5, exchange occurs by and OH -ion-catalysed paths, and the results provide further evidence for associative substitution mechanisms with l. New direct measurements of the rate of the Dushman reaction, at [I"]< 10 mol F have been obtained and confirm the importance of the polymerization of HIO3 in... 

Iodine.— The exchange of oxygen atoms between iodic acid and water has been followed by using 0 as a tracer, using a rapid chemical quenching technique. The rate law at 5 °C and 1 mol dm ionic strength has the form (28). The term... 

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