Enzyme reactions rapid flow techniques

A stable enzyme-D-glucose intermediate has been obtained in the hydrolysis of methyl a-D-[ C]glucopyranoside by an a-D-glucosidase from Saccharomyces oviformis Phenol was used to terminate the reaction and to trap the intermediate in a rapid-flow technique. The intermediate appears to be covalently linked, since continuous washing of the denatured protein failed to remove the radiolabel, which was also retained by a tryptic peptide isolated by gel filtration. Treatment of the intermediate with acid released D-[ C]glucose. The radiolabel was not bound when the enzyme was replaced with bovine albumin or when jS-D-glucosylamine (a potent inhibitor of a-D-glucosidase) was added with the enzyme. 

Enzyme linked electrochemical techniques can be carried out in two basic manners. In the first approach the enzyme is immobilized at the electrode. A second approach is to use a hydrodynamic technique, such as flow injection analysis (FIAEC) or liquid chromatography (LCEC), with the enzyme reaction being either off-line or on-line in a reactor prior to the amperometric detector. Hydrodynamic techniques provide a convenient and efficient method for transporting and mixing the substrate and enzyme, subsequent transport of product to the electrode, and rapid sample turnaround. The kinetics of the enzyme system can also be readily studied using hydrodynamic techniques. Immobilizing the enzyme at the electrode provides a simple system which is amenable to in vivo analysis. 

In order to use the stopped-flow technique, the reaction under study must have a convenient absorbance or fluorescence that can be measured spectrophotometri-cally. Another method, called rapid quench or quench-flow, operates for enzymatic systems having no component (reactant or product) that can be spectrally monitored in real time. The quench-flow is a very finely tuned, computer-controlled machine that is designed to mix enzyme and reactants very rapidly to start the enzymatic reaction, and then quench it after a defined time. The time course of the reaction can then be analyzed by electrophoretic methods. The reaction time currently ranges from about 5 ms to several seconds. 

The concept of ordered interactions of substrates with the enzyme and ordered dissociation of the products was advanced by Koshland in 1954. From then through the 1960s the introduction of stopped-flow techniques and relaxation methods allowed rapid reactions to be followed and the identification of transient intermediates, from which much more complex kinetic analyses have emerged (Fersht,1977). 

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... 

Pre-steady-state stopped-flow and rapid quench techniques applied to Mo nitrogenase have provided powerful approaches to the study of this complex enzyme. These studies of Klebsiella pneumoniae Mo nitrogenase showed that a pre-steady-state burst in ATP hydrolysis accompanied electron transfer from the Fe protein to the MoFe protein, and that during the reduction of N2 an enzyme-bound dinitrogen hydride was formed, which under denaturing conditions could be trapped as hydrazine. A comprehensive model developed from a computer simulation of the kinetics of these reactions and the kinetics of the pre-steady-state rates of product formation (H2, NH3) led to the formulation of Scheme 1, the Thorneley and Lowe scheme (50) for nitrogenase function. 

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. 

When the rate of a reaction is faster than the time it takes us to introduce and mix reagents, we must turn to a method that achieves rapid mixing and has the ability to monitor the extent of reaction at various times. The most common methods involve flow tubes (Figure 7.16). The reactants are mixed at the point where the two tubes intersect, and then theex-tent of reaction is followed by an analysis of the mixture at various points along the observation tube. The flow rate of the tube is known, so that the time from the point of mixing is known at each analysis point along the tube. In the stopped-flow method, the flow is suddenly stopped at various times, and the analysis is performed at the same point in the observation tube at each different time. Several commercial versions of this apparatus are available for stopped-flow analyses, and it is a particularly common method for the analysis of enzyme-catalyzed reactions. With this technique reactions with half-lives as short as milliseconds can be measured. 

The steady-state and rapid equilibrium kinetics do not give detailed information on the existence of multiple intermediates or on their lifetimes. Such information is provided by fast (or transient) kinetics. The methods can be divided in two categories rapid-mixing techniques (stopped-flow, rapid-scanning stopped-flow, quenched flow) which operate in a millisecond time scale and relaxation techniques (temperature jump, pressure jump) which monitor a transient reaction in a microsecond time scale. Most of the transient kinetic methods rely on spectrophotomet-rically observable substrate changes during the course of enzyme catalysis. 

Measurements of reactions that occur in less than a few seconds require special techniques to speed up the mixing of the enzyme and substrate. One way to achieve this is to place solutions containing the enzyme and the substrate in two separate syringes. A pneumatic device then is used to inject the contents of both syringes rapidly into a common chamber that resides in a spectrophotometer for measuring the course of the reaction (fig. 7.5). Such an apparatus is referred to as a stopped-flow device because the flow stops abruptly when the movement of the pneumatic driver is arrested. In this type of apparatus it is possible to make kinetic measurements within about 1 ms after mixing of enzyme and substrate. 

Photodissociation of CO ( flash ) from the reduced enzyme after mixing with O2 ( flow ) in the dark has been the main method of initiating the reaction. Some concern has been voiced as to the possibility that this technique might introduce artifacts due to CO, but results using a rapid O2 mixing system without CO have coirfirmed the applicabihty of the flow/flash technique. Yet, it has been shown by infrared spectroscopy that upon photolysis, the heme 03-bound CO is first transferred in less than a picosecond to the nearby Cub, to which it remains bound for about a microsecond at room temperature before diffusing out of the enzyme, as also verified by EXAFS data. ... 

Transient-kinetic techniques most often rely on the rapid mixing of reactants with enzyme to initiate the reaction. This mixing is essential so that all enzyme molecules start reaction in synchrony with one another therefore, the time dependence of the observable reactions dehnes the kinetics of interconversion of enzyme intermediate states. Because mixing requires a hnite amount of time, conventional methods are limited in their ability to measure very fast reactions. For example, a typical value for the dead time of a stopped-flow instrument is approximately 1 ms, which is because of the time it takes to hll the observation cell. Thus, reactions with a half-life of less than 1 ms (rate > 700 s ) are difficult to observe depending on the signal to noise... 

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