Fluorescent Enzyme Cofactors

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There is presently interest in the use of fluorescence from tissues, either from the intrinsic fluorophores or fiom extrinsically added probes. Much of the fluorescence 

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Tissue also contains some endogenous species that exhibit fluorescence, such as aromatic amino acids present in proteins (phenylalanine, tyrosine, and tryptophan), pyridine nucleotide enzyme cofactors (e.g., oxidized nicotinamide adenine dinucleotide, NADH pyridoxal phosphate flavin adenine dinucleotide, FAD), and cross-links between the collagen and the elastin in extracellular matrix.100 These typically possess excitation maxima in the ultraviolet, short natural lifetimes, and low quantum yields (see Table 10.1 for examples), but their characteristics strongly depend on whether they are bound to proteins. Excitation of these molecules would elicit background emission that would contaminate the emission due to implanted sensors, resulting in baseline offsets or even major spectral shifts in extreme cases therefore, it is necessary to carefully select fluorophores for implants. It is also noteworthy that the lifetimes are fairly short, such that use of longer lifetime emitters in sensors would allow lifetime-resolved measurements to extract sensor emission from overriding tissue fluorescence. 

Many enzymes use the flavin cofactor at the active site the fluorescent active site approach can be applied to study these enzymes at the single-molecule level. Other naturally fluorescent enzymes, like those that contain NAD cofactors, can in principle be studied, although the bluer fluorescence of NAD poses a technical challenge for singlemolecule fluorescence detection. As the approach uses the natural fluorescence of the enzyme, no labeling with fluorescent probes is needed, offering no or minimum perturbation on the enzyme structure and function. 

The cofactor appears to include a novel pterin.996-998 The properties of the pterin depend upon the nature of the side-chain in the 6-position. The structure shown in Figure 39 has been proposed997 on the basis that molybdopterin is related to urothione, oxidized to pterin-6-carboxylic acid, and contains in the side-chain two sulfur groups, a double bond, a hydroxyl function and a terminal phosphate group. Two stable fluorescent derivatives of molybdopterin have been characterized,999 which may be of value in view of the extreme instability of the native molybdoprotein when released from the enzyme. 

The addition of 1 x 10 3 M acetyl glutamate to frog liver enzyme preparation causes a 10% and immediate increase in fluorescence. This effect has been confirmed by Edelhoch (8), who concurs in our interpretation that this finding signifies acetyl glutamate-induced conformational changes of carbamyl-P synthetase. We believe that the decrease in stability induced by substrates and cofactors is, whenever it is found to occur, the simplest and most sensitive method available for the detection and study of conformational changes in enzymic proteins. 

This mechanistic interpretation of the structure is supported by the results from site-directed mutagenesis, where Arg A301 was replaced relatively conservatively by a Lys (Lancaster etal., 2001). Strain FrdA-R301K contained a variant enzyme, very similar to the wild-type enzyme in terms of cofactor and subunit composition, in particular a fluorescence typical... 

The determination of the quantity of protein bound to the insoluble carrier sometimes causes difficulties. The methods usually applied are laborious or somewhat inaccurate. Labeling of assayed protein, for instance with C-acet-anhydride, makes it possible to carry out a very fast and exact determination of immobilized protein The determination of bound enzyme C-labeled aldolase after its immobilization on polyacrylamide can serve as an example The concentration measurements of certain proteins are based on their ability to bind certain ligands. Radiolabels such as or H-biotin have been used for the determination of avidin by direct binding or for biotin assay by isotopic dilution Cofactor and fluorescent labeled ligands have been also used for the monitoring of specific protein binding reactions. 

The characteristic fluorescence spectra of oxidized pterins observed in solutions of denatured molybdenum enzymes provided some of the first clues that the molybdenum cofactor contains a pterin unit (24). 

Evidence for the presence of a pterin cofactor in this enzyme was obtained by Mukund and Adams (282) the fluorescence spectrum of the... 

The idea to analyze the catalysis by a single enzyme molecule was an obvious step. This was first demonstrated by Peter Lu and Sunney Xie [38]. They were able to show intensity fluctuation of the flavin cofactor of cholesterol oxidase using confocal single molecule detection. Our idea of single enzyme catalysis was to follow the turnover of a substrate into the product by catalysis of a single enz une molecule. We chose horse radish peroxidase and a non-fluorescence substrate dihydro-rhodamine which is turned over in the fluorescent product... 

Many enzymes use organic cofactors, such as flavin and porphyrin, at the active sites for catalysis. Some of these organic cofactors, especially flavin, have intrinsic fluorescence, and can be imaged readily at the singlemolecule level. If the fluorescence of these cofactors is coupled with the state of the active site in the catalytic cycle, monitoring the fluorescence of the active site can directly probe the catalysis. The classic example of this... 

Chemiluminescence offers a useful alternative to both fluorescence and radioactivity, as it does not require an excitation source, there is less light scattering, and the problem of source instability is absent. The radiation hazard is also absent from chemiluminescent molecules. Most chemiluminescent reagents and their conjugates are stable and can be applied to both homogeneous and heterogenous assays. The chemiluminescent label may be attached to an enzyme or may serve either as a substrate or as a cofactor for the enzyme. 

The GFP from the jellyfish Aequorea victoria, although not an enzyme, has become widely used as a marker for gene expression and localization. Although the fluorophore of GFP is not technically a protein-derived cofactor, it is a protein-derived fluorophore. This is another example of posttranslational modifications, which endow amino acid residues with a new function. In this case, the new function is not one which assists in catalysis. Instead, the results of these posttranslational modifications create new fluorescent properties, which serve a different biological function. As with most of the protein-derived cofactors discussed earlier, the presence and identity of the fluorophore is not evident from the amino acid sequence of the protein. The structure of the GFP fluorophore and mechanism of its biosynthesis were deduced from structural analyses. The X-ray crystal structure of GFP revealed that the covalently bound fluorescent chromophore is derived from three adjacent amino acids, serine-tyrosine-glycine on the polypeptide chain (Figure 13). ... 

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