Enzymatic turnover

Sharma and Reed, 1976)]. In proteins the coordination number 4 is most common, where the zinc ion is typically coordinated in tetrahedral or distorted tetrahedral fashion. The coordination polyhedron of structural zinc is dominated by cysteine thiolates, and the metal ion is typically sequestered from solvent by its molecular environment the coordination polyhedron of catalytic zinc is dominated by histidine ligands, and the metal ion is exposed to bulk solvent and typically binds a solvent molecule (Vallee and Auld, 1990). The inner-sphere coordination number of catalytic zinc may increase to 5 during the course of enzymatic turnover, and several five-coordinate zinc enzyme—substrate, enzyme product, and enzyme-inhibitor complexes have been studied by high-resolution X-ray crystallographic methods (reviewed by Matthews, 1988 Christianson and Lipscomb, 1989). The coordination polyhedron of zinc in five coordinate examples may tend toward either trigonal bipyramid or octahedral-minus-one geometry. 

It is well known that the O2 reduction site of bovine heart cytochrome c oxidase in the fuUy oxidized state exhibits variable reactivity to cyanide and ferrocytochrome c, which is dependent on the method of purihcation (Moody, 1996). Some preparations react with cyanide extremely slowly at an almost immeasurable rate and are known as the slow form. Other preparations, which react at a half-Ufe of about 30 s, are known as the fast form (Brandt et al., 1989). Electronic absorption spectra of the slow-and fast-form preparations exhibit Soret bands at 418 and 424 nm, respectively. The two forms often coexist in a single preparation (Baker et al., 1987). Both forms exhibit an identical visible-Soret spectrum in the fully reduced state. The slow-form preparation can be converted to the fast form by dithionite reduction followed by reoxidation with O2. The fast form thus obtained returns to the slow form spontaneously at a rate much slower than the enzymatic turnover rate. Thus, the slow form is unlikely to be involved in the enzymatic turnover (Antoniniei a/., 1977). It should be noted that no clear experimental evidence has been reported for direct involvement of the fast form in the enzyme turnover, although its direct involvement has been widely accepted. The third species of the fully oxidized O2 reduction site, which appears in the partially reduced enzyme, reacts with cyanide 10 —10 times more rapidly than the fast form (Jones et al., 1984). In the absence of a reducing system, no interconversion is detectable between the slow and the fast forms (Brandt et al., 1989). Thus, the heterogeneity is expected to inhibit the crystallization of this enzyme. In fact, the enzyme preparations providing crystals showing X-ray diffraction at atomic resolution are the fast form preparation. 

If it is not possible to obtain an end point or complete conversion to product, substrate concentrations can be determined from the rate dependence of the enzyme reaction under conditions where [S] KM (Sect. 8.2.2). The sensitivity of the method is not as great as can be achieved by using equilibrium end point measurements, since accuracy depends on analysis of the initial rate of enzymatic turnover. 

When more than one kinetic step is rate limiting in an enzymatic turnover, the reaction is said to be kinetically complex. 

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]...

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... 

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... 

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...

Figure 7. Liposome-assisted catalysis. (A) Dependency of the initial hydrolysis rate of C16-O Np (nitrophenyl-pamitate) catalyzed by 1 mM carbobenzoxy-Phe-His-Leu-OH on the substrate concentration, in 0.05 M borate buffer pH 8.5. The filled circles are relative to the self-hydrolysis (no peptide, no liposomes). Open triangles are without liposomes, open squares with liposomes. (B) The pseudo-enzymatic turnover of the catalytically active liposomes. The catalytic activity results primarily from the binding (and solubilization) of a very hydrophobic histidin-containing peptide and the very hydrophobic substrate.

Hardeland, U., Steinacher, R., Jiricny, J., and Schar, P. (2002). Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover. EMBOJ 21, 1456-1464. 

Glucose-6-phosphate is a feedback inhibitor of the enzyme hexokinase and causes a decrease in the enzymatic turnover rate (activity) of that enzyme. 

---