Single- molecule enzymatic turnover

Like enzymology in general, single-molecule enzymology is primarily limited by assay developments. Here, I list the single-molecule enzymatic turnover assays by fluorescence detection using... 

In 1998, we reported the real-time observation of enzymatic turnovers of a single-molecule cholesterol oxidase, a flavoenz3mie that catalyzes oxidation of cholesterol by oxygen [15] (Fig. 22.lA). The active site of the enz3une, flavin adenine dinucleotide (FAD), (Fig. 22.IB), is naturally fluorescent in its oxidized form but not in its reduced form. With excess amounts of cholesterol... 

Fig. 22.1. (A) Enzymatic cycle of cholesterol oxidase which catalyzes the oxidation of cholesterol by oxygen. The enzyme s naturally fluorescent FAD active site is first reduced by a cholesterol substrate molecule, generating a non-fluorescent FADH2, which is then oxidized by oxygen. (B) Structure of FAD, the active site of cholesterol oxidase. (C) A portion of the fluorescence intensity time trace of a single cholesterol oxidase molecule. Each on-off cycle of emission corresponds to an enzymatic turnover. (D) Distribution of emission on-times derived from (C). The solid line is the convolution of two exponential functions with rate constants fci[S] = 2.5 s and fc2 = 15.3 s, reflecting the existence of an intermediate, ES, the enzyme-substrate complex, as shown in the kinetic scheme in the inset. From ref. [15]...

Fig. 24.2. Single-molecule recording of T4 lysozyme conformational motions and enzymatic reaction turnovers of hydrolysis of an E. coli B cell wall in real time, (a) This panel shows a pair of trajectories from a fluorescence donor tetramethyl-rhodamine blue) and acceptor Texas Red (red) pair in a single-T4 lysozyme in the presence of E. coli cells of 2.5mg/mL at pH 7.2 buffer. Anticorrelated fluctuation features are evident. (b) The correlation functions (C (t)) of donor ( A/a (0) Aid (f)), blue), acceptor ((A/a (0) A/a (t)), red), and donor-acceptor cross-correlation function ((A/d (0) A/d (t)), black), deduced from the single-molecule trajectories in (a). They are fitted with the same decay rate constant of 180 40s. A long decay component of 10 2s is also evident in each autocorrelation function. The first data point (not shown) of each correlation function contains the contribution from the measurement noise and fluctuations faster than the time resolution. The correlation functions are normalized, and the (A/a (0) A/a (t)) is presented with a shift on the y axis to enhance the view, (c) A pair of fluorescence trajectories from a donor (blue) and acceptor (red) pair in a T4 lysozyme protein without substrates present. The acceptor was photo-bleached at about 8.5 s. (d) The correlation functions (C(t)) of donor ((A/d (0) A/d (t)), blue), acceptor ((A/a (0) A/a (t)), red) derived from the trajectories in (c). The autocorrelation function only shows a spike at t = 0 and drops to zero at t > 0, which indicates that only uncorrelated measurement noise and fluctuation faster than the time resolution recorded (Adapted with permission from [12]. Copyright 2003 American Chemical Society)...

Single-molecule spFRET fluorescence trajectories contain detailed information about the conformational motion associated with the enzymatic turnovers. The upper panel in Fig. 24.4 shows an expanded portion of a trajectory (middle panel) recorded from donor fluorescence of a single-pair donor-acceptor labeled protein with substrate present. By comparison, the lower panel shows a portion of a donor-fluorescence trajectory recorded from a donor-only labeled T4 lysozyme under the same conditions. The... 

E-fS ES ES. The standard deviation of the distribution, (Atopen ) = 8.3 2ms, reflects the distribution bandwidth. For the individual T4 lysozyme molecules examined under the same enz unatic reaction conditions, we found that the first and second moments of the single-molecule topen distributions are homogeneous, within the error bars. The hinge-bending motion allows sufficient structural flexibility for the enzyme to optimize its domain conformation the donor fluorescence essentially reaches the same intensity in each turnover, reflecting the domain conformation reoccurrence. The distribution with a defined first moment and second moment shows typical oscillatory conformational motions. The nonequilibrium conformational motions in forming the active enzymatic reaction intermediate states intrinsically define a recurrence of the essentially similar potential surface for the enzymatic reaction to occur, which represents a memory effect in the enzymatic reaction conformational dynamics [12,41,42]. 

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. 14. A biphasic plot to determine and for the oxidation of Vivid Red by CYP3A4 immobilised on a microarray surface. The microarray-generated kinetic data set for the turnover of Vivid Red by a CYP3A4-BCCP P0R-BCCP complex (Fig. 13) was fitted to a biphasic, multlsite model using non-linear regression.Tlie resultant catalytic parameters, and describe the enzymatic activity when only a single molecule ot substrate is bound in the active site at any one time these values conform well with literature values obtained with baculosome preparations ot CYP3A4.

Km for an enzymatic reaction are of significant interest in the study of cellular chemistry. From equation 13.19 we see that Vmax provides a means for determining the rate constant 2- For enzymes that follow the mechanism shown in reaction 13.15, 2 is equivalent to the enzyme s turnover number, kcat- The turnover number is the maximum number of substrate molecules converted to product by a single active site on the enzyme, per unit time. Thus, the turnover number provides a direct indication of the catalytic efficiency of an enzyme s active site. The Michaelis constant, Km, is significant because it provides an estimate of the substrate s intracellular concentration. 

This means that y/x substrate molecules are converted to product molecules every second. Correspondingly, the time required for a single conversion is x/y seconds. Turnover numbers for most enzymes usually range from 1 to 1 O per second. A few enzymes have turnover numbers above 10 (Table 6-1). The ability of a cell to produce a given amount of product by an enzymatic reaction during its life span is proportional to the turnover number and the number of molecules of that enzyme in the cell. Because turnover numbers... 

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