Single turnover events

To substantiate the initiating species, reaction of excess benzyl alcohol with one equivalent of DMAP to lactide was investigated to generate a single turnover event. The lactide was ring opened with the formation of the benzyl ester, however due to the large excess of the benzyl alcohol and DMAP catalyst, transesterification with the diester product, afforded the monoester . Conversely, 5ec-phenethyl alcohol ring-opened lactide quantitatively to the diester product with no evidence of adverse side... 

The most useful approaches for obtaining information regarding the existence of intermediates and their lifetimes are fast reaction methods that mix enzyme and substrate within milliseconds, which permits the observation of single turnover events by various spectroscopic methods. Alternatively the reaction is rapidly quenched at known time intervals and its progress is analyzed chromatographically. In many cases in which an intermediate accumulates to the level of the enzyme concentration, such methods reveal the presence of burst kinetic that feature the rapid buildup of the intermediate in the transient phase followed by its slower rate of formation/decay in the steady state. The simplest kinetic scheme consistent with this phenomenon is given by... 

Lakes with two turnover events per year are dimictic. Monomictic lakes may have a single turnover event, while other lakes described as polymictic experience multiple episodes of weaker stratification and turnover events. Some permanently ice covered lakes never turn over, are amictic and maintain isothermal temperatures due to a lack of wind induced mixing. At times of calm, stable weather during the period of... 

Venturole G and Melandri BA (1982) The localized coupling of bacterial photophosphorylation. Effect of Antimycin a and N,N-dicyclohexylcarbodiimide in chromatophores from Rhodopseudomonas sphaeroides, Ga, studied by single turnover event analysis, Biochim. Biophys. Acta 637, 447-456. 

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... 

Fig. 25.1. Analysis of the catalytic activity of CalB at the single-moiecuie ievei. (a) Detection of single enzymatic turnover events of the enzyme CaiB. The fluorogenic substrate BCECF-AM is hydrolyzed by CalB yielding the highly fluorescent dye BCECF. (b) Proposed reaction scheme explaining dynamic disorder. The enzyme interconverts between different conformations with the rate constants Oa, b. Each conformation hydrolyzes the substrate with its own rate constant fci. If conformational changes are slower than the catalytic reaction, a certain conformation performs several turnover cycles before it switches into another conformation. While subsequent turnovers in one conformation are correlated, the system loses its memory after a conformational change...

Fig. 25.2. Analysis of the catalytic activity and the inactivation of a-chymotrypsin at the single-molecule level, (a) Detection of single enzymatic turnover events of a-chymotrpysin. The fluorogenic substrate (suc-AAPF)2-rhodamine 110 is hydrolyzed by a-chymotrypsin, yielding the highly fluorescent dye rhodamine 110. (b) Representative intensity time trace for an individual a-chymotrypsin molecule undergoing spontaneous inactivation imder reaction conditions, (c) Inactivation trace for the intensity time transient in (b), obtained by counting the amount of turnover peaks in (b) in 10 s intervals. After approximately 1000 s, the enzyme deactivates through a transient phase with discrete active and inactive states, (d) Proposed model for the inactivation process. An initial active state is in equilibrium with an inactive state. This inactive state converts to another inactive state irreversibly whereby the corresponding active state has a lower activity than the previous one. All the transitions involved have energy barriers that can be overcome spontaneously at room temperature...

With single-turnover selection processes, only a single catalytic event is necessary for selection therefore, the selected variants are not necessarily improved catalysts. 

For the purpose of the present discussion the term transient kinetics is applied to the time course of a reaction from the moment when enzyme and substrate are mixed, t=0, until either a steady state or equilibrium is established. The difference between the kinetic problems discussed in section 3.3 and in the present section is, respectively, the presence of catalytic as distinct from catalytic concentrations of enzyme. Here we are concerned with the stoichiometry of enzyme states. Transient kinetic experiments with enzymes can be divided into two types. The first of these (multiple turnover) is carried out under the condition that the initial concentrations of substrate and enzyme are Cs(0) Ce(0) and c it) can, therefore, be regarded as constant throughout the course of the reaction until a steady state is attained. Alternatively, in a single turnover reaction, when Cs(0)reaction intermediates is observed until the overall process is essentially complete. These two possibilities will be illustrated with specific examples. In connection with a discussion of the approach to the steady state, in section 3.3 it was emphasized that, at t = 0, the concentrations of the intermediates, enzyme-substrate and enzyme-product complexes, are zero and, therefore, the rate of product formation is also zero. Under the experimental conditions used for steady state rate measurements and for enzyme assays, the first few seconds after the initiation of a reaction are ignored. However, when the experimental techniques and interpretation discussed below are used, events during the first few milliseconds of a reaction can be analysed and provide important information. With suitable monitors it is possible to follow the formation and decay of enzyme complexes with substrates and... 

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