Turnover of enzymes

An attractive strategy for predicting the clinical significance of irreversible inhibition is to use human hepatocytes wherein the natural turnover of enzymes might be preserved and in vivo cellular concentrations of inhibitors and metabolites would be achieved. Zhao et al. demonstrated time-dependent inactivation of CYP3A in cryopreserved hepatocytes for amprenavir, diltiazem, erythromycin, raloxifene, and TAO (126). Except for TAO, significant differences in inactivation efficiency potency between hepatocytes and HLMs were... 

Measurement of luciferase activity. This was conducted by the following two methods 1. Luciferase activity was determined by a flavin injection assay in which 500 pL of photoreduced flavin mononucleotide were rapidly mixed with 500 pL of a solution containing luciferase, tetradecanal (4.7 mM) and 20 mM of phosphate buffer at pH 7.0 2. NADH or DTT was added to the cuvette, containing luciferase, tetradecanal and phosphate buffer and FMN. Under these conditions long-lived luminescence stimulated by multiple turnovers of enzyme was observed. 

Nonelectrogenic functions. During the resting state, there is a continual slow release of isolated quanta of the transmitter that produces electrical responses at the postjunctional membrane [miniature end-plate potentials (mepps)] that are associated with the maintenance of physiological responsiveness of the effector organ. A low level of spontaneous activity within the motor units of skeletal muscle is particularly important because skeletal muscle lacks inherent tone. The activity and turnover of enzymes involved in the synthesis and inactivation of neurotransmitters, the density of presynaptic and postsynaptic receptors, and other characteristics of synapses probably are controlled by trophic actions of neurotransmitters or other trophic factors released by the neuron or the target cells. 

The minimum number of turnovers of enzyme before complete inactivation occurred was calculated to be 10,000. It took 5,000 turnovers of enzyme for complete inactivation of Neurospora PPO (Dletler and Lerch, quoted by Solomon, 1981). 

Kinetic identification of reaction intermediates Singk ami multiple turnover of enzyme reactions... 

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. 

Li, Z., and Meighen, E. A. (1994). The turnover of bacterial luciferase is limited by a slow decomposition of the ternary enzyme-product complex of luciferase, FMN, and fatty acid. J. Biol. Chem. 269 6640-6644. 

In be complexes bci complexes of mitochondria and bacteria and b f complexes of chloroplasts), the catalytic domain of the Rieske protein corresponding to the isolated water-soluble fragments that have been crystallized is anchored to the rest of the complex (in particular, cytochrome b) by a long (37 residues in bovine heart bci complex) transmembrane helix acting as a membrane anchor (41, 42). The great length of the transmembrane helix is due to the fact that the helix stretches across the bci complex dimer and that the catalytic domain of the Rieske protein is swapped between the monomers, that is, the transmembrane helix interacts with one monomer and the catalytic domain with the other monomer. The connection between the membrane anchor and the catalytic domain is formed by a 12-residue flexible linker that allows for movement of the catalytic domain during the turnover of the enzyme (Fig. 8a see Section VII). Three different positional states of the catalytic domain of the Rieske protein have been observed in different crystal forms (Fig. 8b) (41, 42) ... 

X-ray structures of mitochondrial 6ci complexes from three different sources (113, 124, 125) have found the b- and c-type hemes at roughly identical positions, whereas the Rieske protein was seen in different places as a function of crystal space group and presence or absence of inhibitors of the enzyme. This fact was interpreted to suggest a long-range conformational movement of the Rieske protein during turnover of the complex. The range of observed positions of the Rieske protein indicated that the soluble domain can move like a... 

There is no evidence of a general overactivity in DA function in schizophrenic patients. Plasma prolactin is not reduced, so the DA inhibitory control of its release is normal there is no recorded increase in DA turnover as CSF and plasma levels of its major metabolite HVA are normal and dyskinesias, which would reflect increased DA activity, are rare. PM studies have shown no consistent increases in DA brain levels, although some reports show an increase in the left amygdala, or in the activity of enzymes involved in its synthesis (tyrosine hydroxylase) or metabolism (MAO). For a review of the neurochemistry see Reynolds (1995). 

The fructose-specific PTS in R. sphaeroides is simpler than the one in E. coli or S. typhimurium in that it consists of only two proteins. Besides the fructose specific ll , a class II enzyme, there is only one cytoplasmic component called soluble factor (SF) [48]. We now know that SF consists of IIl , HPr and E-I covalently linked [109]. 11 and SF form a membrane-bound complex whose association-dissociation dynamics is much slower than the turnover of the system. Therefore, the complex is the actual catalytic unit in the overall reaction and P-enolpyruvate is the direct phosphoryl group donor [102],... 

Details of the mechanism of naphthalene dioxygenase during a single turnover of the enzyme have been revealed, and conhrmed the separate roles of the dioxygenase and the ferredoxin electron transfer protein. This made it possible to propose a reaction cycle for the reaction (Wolfe et al. 2001). 

They have an exceedingly high specific activity per active site the turnover number y is as high as 10 to 10 s in certain enzyme reactions, while at ordinary electrocatalysts having a number of reaction sites on the order of 10 cm , yhas a value of about 1 s at a current density of lOmA/cm. Thus, the specific catalytic activity of tfie active sites of enzymes is many orders of magnitude fiigher tfian tfiat of all other known catalysts for electrochemical (and also chemical) processes. 

Chemo-enzymatic epoxidation of unsaturated fatty acids with aqueous H2O2 has been conducted with considerable success and here we have a remarkable situation that undesirable ring opening of the epoxide does not occur. Excellent activity and stability has been realized with Novozym 435, a Candida antartica lipase B immobilized on polyacryl. This enzyme is readily separable, can be used several times without loss of activity, and has a turnover of more than 2,00,000 moles of products per mole of catalyst (Bierman et al., 2000). 

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. 

The examples above serve to illustrate that the conformational dynamics of enzyme turnover create multiple, specific binding pocket configurations throughout the reaction pathway, each representing a distinct opportunity for drug binding and inhibition. 

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