Enzyme turnover rate

The chemical shift of the phosphorus resonance of various nucleotides has been studied as a function of pH in the presence and absence of RNase A. The signal shifts upheld on protonation of the phosphate and the apparent pATa of the phosphate group in 2 -CMP complex with RNase is the same as the pATa of histidine-119 in this enzyme as determined by n.m.r. From n.m.r. relaxation rates for the ternary complex manganese(n)-phosphate-E. coli alkaline phosphatase, it has been concluded that an outer-sphere complex is formed which has a shorter lifetime than the enzyme turnover rate. The latter conclusion is consistent with the participation of the complex in the enzymic reaction. 

Diffusion barriers are typically used where the chemical reaction responsible for sensing is slow relative to the diffusion rate of the analyte to the active portion of the sensor. As shown above, a gas-permeable membrane can act as a diffusion barrier in some cases for gas sensors. Diffusion barriers are also often used in amperometric enzyme sensors which exploit the native selectivity of an enzyme-substrate reaction to measure the concentration of the substrate. If the substrate is at a high concentration, the enzyme may become saturated, especially if the enzyme turnover rate is low. In this condition, the signal will plateau and no longer be dependent upon the substrate concentration. A diffusion barrier between the enzyme layer and the sample reduces the flux of the substrate to the enzyme and, thus, prevents saturation of the enzyme and increases the linear range of the sensor. 

Figure 8.58 Schematic illustration of reaction coordinate diagram of Triose Phosphate Isomerase (TIM) enzyme illustrating near perfect energy landscape pathway allowing for near perfect 1 1 1 stoichiometric equilibrium between all enzyme-bound species optimal for flux through from one enzyme-bound species to another. Enzyme turnover rate kobs is at the diffusion limit, the rate determining step is the association of dihydroxy acetone phosphate (DHAP) with the TIM catalytic site, see Fig. 8.1, hence chemistry is not rate limiting. Therefore, TIM is considered a perfect enzyme For TIM enzyme assay see Fig. 8.17 for TIM enzyme mechanism see Fig. 8.49 (illustration adapted from Knowles, 1991, Fig. 2).

Enzyme turnover rate (iJimol substrate Liver 1.94... 

Despite enormous progress in understanding Bi2-mediated reactions, several seminal questions remain. Two issues of great interest are the molecular mechanism of cobalt-carbon bond activation in coenzyme B12 and the function of the trans axial ligand in catalysis. While the cobalt-carbon bond of coenzyme B12 is relatively weak, it must be further activated by a factor of two (in terms of BDE) upon enzyme complexation to yield bond homolysis rates consistent with enzyme turnover rates. The role of the trans axial base has received a great deal of attention. The novel... 

The advantage of studying enzymes is that they can be regenerated by known substrates. Although they might be influenced by the enzyme turnover rate, the enzymatic substrate can sustain a level of activity that is harder to find in other interesting biological systems. 

Hull, W. E., Halford, S. E., Gutfreund, H. Sykes, B. D. (1976). P nuclear magnetic resonance study of alkaline phosphatase the role of inorganic phosphate in limiting the enzyme turnover rate at alkaline pH. Biochemistry, 15, 1547-61. 

Enzymes are excellent catalysts for two reasons great specificity and high turnover rates. With but few exceptions, all reac tions in biological systems are catalyzed by enzymes, and each enzyme usually catalyzes only one reaction. For most of the important enzymes and other proteins, the amino-acid sequences and three-dimensional structures have been determined. When the molecular struc ture of an enzyme is known, a precise molecular weight could be used to state concentration in molar units. However, the amount is usually expressed in terms of catalytic activity because some of the enzyme may be denatured or otherwise inactive. An international unit (lU) of an enzyme is defined as the amount capable of producing one micromole of its reaction product in one minute under its optimal (or some defined) reaction conditions. Specific activity, the activity per unit mass, is an index of enzyme purity. 

In this cycle, step 3, the dissociation of the two proteins, is rate determining and is rate determining in the overall enzyme turnover. 

ALASl. This repression-derepression mechanism is depicted diagrammatically in Figure 32-9. Thus, the rate of synthesis of ALASl increases greatly in the absence of heme and is diminished in its ptesence. The turnover rate of ALASl in rat liver is normally rapid (half-life about 1 hour), a common feature of an enzyme catalyzing a rate-limiting reaction. Heme also affects translation of the enzyme and its transfer from the cytosol to the mitochondrion. 

After secretion from the cell, certain lysyl residues of tropoelastin are oxidatively deaminated to aldehydes by lysyl oxidase, the same enzyme involved in this process in collagen. However, the major cross-links formed in elastin are the desmosines, which result from the condensation of three of these lysine-derived aldehydes with an unmodified lysine to form a tetrafunctional cross-hnk unique to elastin. Once cross-linked in its mature, extracellular form, elastin is highly insoluble and extremely stable and has a very low turnover rate. Elastin exhibits a variety of random coil conformations that permit the protein to stretch and subsequently recoil during the performance of its physiologic functions. 

The turnover rate of a transmitter can be calculated from measurement of either the rate at which it is synthesised or the rate at which it is lost from the endogenous store. Transmitter synthesis can be monitored by administering [ H]- or [ " C]-labelled precursors in vivo these are eventually taken up by neurons and converted into radiolabelled product (the transmitter). The rate of accumulation of the radiolabelled transmitter can be used to estimate its synthesis rate. Obviously, the choice of precursor is determined by the rate-limiting step in the synthetic pathway for instance, when measuring catecholamine turnover, tyrosine must be used instead of /-DOPA which bypasses the rate-limiting enzyme, tyrosine hydroxylase. 

Important inherent characteristics of an enzyme that should be considered are the substrate affinity, characterized by the Michaelis constant the rate of turnover fecat> providing the catalytic efficiency fecat/ M. and the catalytic potential. Several attempts to compare enzyme catalysis with that of platinum have been published. Direct comparisons are difficult, because enzyme electrodes must be operated in aqueous electrolyte containing dissolved substrate, whereas precious metal electrodes aie often supplied with a humidified gaseous stream of fuel or oxidant, and produce water as steam. It is not straightforward to compare tme optimal turnover rates per active site, as it is often unclear how many active sites are being engaged in a film of enzyme on an electrode. 

Many bacteria possess a high-affinity urea uptake system accompanied by an intracellular urease enzyme (135). Nielsen et al. (136) have measured both natural urea turnover rates and gross N mineralization rates in situ using a new... 

The enzymatic activities of intercalated GOx-AM P layered nanocomposites at various pH values and temperatures were compared with the native enzyme in aqueous solution. In both cases, characteristic linear plots consistent with Michalis-Menton kinetics were obtained. The Lineweaver-Burk plots indicated that the reaction rates (Vmax) for free and intercalated GOx (3.3 and 4.0 pM min 1 respectively), were comparable, suggesting that the turnover rate at substrate saturation was only marginally influenced by entrapment between the re-assembled organoclay sheets. However, the dissociation constant (Km) associated with the activity of the enzyme was higher for intercalated GOx (6.63 mM) compared to native GOx (2.94 mM), suggesting... 

Experiments were also carried out injecting [3 -3H]xylosyl-MTA. The results indicated that the molecule has a very low turnover rate in D. verrucosa, since 96% of the recovered radioactivity after 24 h was associated with xylosyl-MTA. Accordingly, it was observed [126] that xylosyl-MTA is resistant to the enzyme MTA-phosphorylase which cleaves MTA but not the xylose analog, which therefore accumulates in the animal. Since xylosyl-MTA is mainly concentrated in the hermaphrodite gland of D. verrucosa and is very abundant in the eggmasses [103], it may play a role in the reproductive biology of D. verrucosa. 

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