tailieunhanh - ENZYME KINETICS A MODERN APPROACH – PART 7

Như đã nói ở trên, độ cong lên trong giai đoạn đầu của phản ứng được đưa ra bởi thời hạn theo cấp số nhân trong Eq. (11,11). Khi thời gian đủ dài, thuật ngữ theo cấp số nhân trở nên không đáng kể nhỏ, và đường cong cơ bản trở thành một đường thẳng. | RELAXATION TECHNIQUES 135 Late Stages of the Reaction As discussed above the upward curvature during the early stages of the reaction is given by the exponential term in Eq. . When time is sufficiently long the exponential term becomes negligibly small and the curve becomes essentially a straight line. For the case S0 Km as t TO Eq. reduces to k2 ET Pt - P0 MEt t - k1 S0 A plot of Pt P0 versus time yields a straight line with slope k2 ET The x-axis intercept of this line sometimes referred to as the relaxation time t Fig. at Pt P0 0 corresponds to 1 t k1 S0 Thus from knowledge of the values of the slope x -intercept and initial substrate concentration estimates of k1 and k2 and be obtained. An estimate of k 1 can be obtained from knowledge of Km k1 and k2 k 1 k1Km k2 This exercise is merely one example among many of pre-steady-state kinetic analysis of enzyme-catalyzed reactions. RELAXATION TECHNIQUES The time resolution of rapid-flow methods is limited by the rate at which two reactants are mixed which is restricted to about 1 ms. To measure faster reactions alternative methods are required. A generally applicable method is the measurement of system adjustment following a relatively small perturbation. A system at equilibrium is perturbed by a sudden temperature or pressure jump applied as a single rapid change or as a periodic oscillation. Changes in the concentration of reactants and products are subsequently monitored. From the patterns observed individual rate constants can be obtained. 136 TRANSIENT PHASES OF ENZYMATIC REACTIONS Consider the opposing reaction k1 A B k-1 11-19 After a rapid perturbation that causes a small disturbance of the equilibrium state of a reaction the change in concentration of either species follows a simple exponential pattern. As discussed in Chapter 1 Eq. describes changes in the concentration of B d B _ _ _ . k1 A - k-1 B k1 A0 - B - k-1 B d t The deviation of B from