TRANSIENT PHASES OF ENZYMATIC REACTIONS

Consider a typical mechanism for an enzyme-catalyzed reaction  

Steady-state kinetic analysis provides estimates of and V ax, where 

To determine individual rate constants (i.e., k and k-i) for the mechanism depicted above, it is necessary to monitor the progress of the reaction before establishment of the steady state. This pre-steady-state region of an enzymatic reaction is called the transient phase of an enzymatic reaction. For this purpose, it is necessary to carry measurements of a single turnover of substrate into product, usually using enzyme concentrations in the range of those of substrate ([E] [S]). 

Two methods exist for the determination of individual rate constants of an enzymatic reaction rapid-reaction techniques and relaxation techniques. In rapid-reaction techniques, reaction rates are determined after 

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This book starts with a review of the tools and techniques used in kinetic analysis, followed by a short chapter entitled How Do Enzymes Work , embodying the philosophy of the book. Characterization of enzyme activity reversible and irreversible inhibition pH effects on enzyme activity multisubstrate, immobilized, interfadal, and allosteric enzyme kinetics transient phases of enzymatic reactions and enzyme... 

Cholinesterase. Cholinesterase can be assayed by determining the choline liberated in the enzymatic reaction by using immobilized choline oxidase. Further, the direct electrochemical registration of thiocholine iodide, the product of the cholinesterase-catalyzed hydrolysis of butyrylthiocholine iodide, has been used [384]. The Glukometer has been adapted to this reaction system by polarizing the platinum electrode to 470 mV versus an Agl electrode in 0.1 M potassium iodide solution. The formation of thiocholine iodide causes an increase in the oxidation current. After a transient phase of about 20 s the reaction rate becomes constant. In the kinetic mode a constant measuring value proportional to the reaction rate is obtained. A... 

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...

Fig. 2 shows incorporation as function of temperature obtained from a crude homogenate of Sulfolobus solfataricus harvested in the late logarithmic growth phase (Fig.l, B). ADP-ribosyl transferase activity increased quickly from 15 C, showed a transient plateau from 30 C to 50°C and reaches a maximum value at 80 °C. To obtain information about cellular distribution of the ADP-ribosyl transferase activity, enzymatic activity was determined either on the soluble protein fraction (14) or on the nucleoprotein fraction obtained according to Searcy (15). The results showed a preferential association of the ADP-ribosyl transferase activity to the nucleoprotein fraction with a 45-fold increase of specific activity relative to cmde homogenate. Identification of the reaction product as ADP-ribose was... 

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