Prosthetic group extraction

Figure 9. MCD spectra of the ferrous pyridine-CO complexes of the extracted prosthetic group of HPII catalase (--), octaethylchlorin (----),...

Figure 2-27. Structure of the reaction center complex of Rps.viridis. a) Whole structure, b) Prosthetic groups extracted from the whole structure (He = heme iron complex).

In the second part, selected immobilized structural and spectroscopic active site models will be discussed and aspects of characterization and analytics of immobilized transition metal complexes will be exemplarily disclosed. Typical techniques include spectroscopic methods addressing the immobilized biomimetic species and determination of metal ion leaching and active site integrity, for example, by selective extraction of the intact biomimetic metal complex - the prosthetic group - from the matrix - the apoenzyme (prosthetic group extraction). The third section gives a short overview of the elementary reaction steps in the catalytic processes and their observation on solid matrixes. Selected immobilized biomimetic functional active site models will be discussed in detail in the last section. 

Figure 20.3 (a) Concept of prosthetic group extraction using Si-0 iinker [22, 23] and (b) compiex types 13-18 successfuiiy reieased. 

In assays of enzyme activities a cofactor, but not a prosthetic group, can be easily lost from the enzyme by dilution during extraction or purification, or removed by agents that will bind the cofactor. For these reasons, an excess of cofactor is routinely added to the assay medium (e.g. in kinase assays) for the measurement of enzyme activity. 

F430, a yellow, water-soluble compound, was first extracted from boiled cells of methanogenic bacteria, a discovery which Wolfe (19) has credited to J. LeGall. Its isolation was first reported by Gunsalus and Wolfe (83). The cofactor has a Soret band in the visible region at 430 nm. Functionally F430 is a prosthetic group of the methylreductase system (24, 84). It is also found in the free state in cell extracts (85). 

Figure 18.5 The glycerol-3-phosphate shuttle. This shuttle is used to bring electrons from cytosolic NADH into mitochondria. The mitochondrial glycerol-3-phosphate dehydrogenase with its FAD prosthetic group is bound to the inner mitochondrial membrane. ETF is electron transfer flavoprotein, which extracts electrons from the FADH2 of mitochondrial glycerol-3-phosphate dehydrogenase and with it reduces ubiquinone (UQ).

This enzyme [also known at l(-[-)-lactate dehydrogenase] was first extracted from bakers yeast by Bernheim in 1928 (272). Bach et al. (273) showed in 1942 that lactate dehydrogenase copurified with a species of cytochrome b, which contained protoheme as prosthetic group. The... 

The sequence of the various steps between uroporphyrinogen III and cobyrinic acid has been the subject of much recent work. A major advance in this area was the observation that sirohydrochlorin (77), the iron-free prosthetic group of the enzyme siroheme, could be modified to accommodate its role as a biosynthetic intermediate. Subsequently a dimethyl isobacteriochlorin (factor II) isolated from P. shermanii was shown to be identical with sirohydrochlorin from E. coli sulphite reductase. The complete stereostructure of (77) was elucidated by a series of biosynthetic experiments using [4- C]- and [5- C]-ALA and independently by more classical structural arguments (B-79MI10401, B-79MI10402>. Sirohydrochlorin labelled biosynthetically from [4- C]ALA and [ CHsJmethionine is incorporated into cobyrinic acid by cell-free extracts of F. shermanii without loss or migration of label. 

The process of streptomycin formation was greatly facilitated by the addition of meat extract or corn steep liquor to the medium. Waksman and coworkers " pointed out that this addition probably provided an organic substance which served as a precursor of the streptomycin molecule as a whole or of an important group in the molecule, or functioned as a prosthetic group in the mechanism essential for the synthesis of streptomycin. They designated such a substance, required for the... 

In 1915, Harden and Norris observed that dried yeast, when mixed with lactic acid, reduced methylene blue and formed pyruvic acid 4). Thirteen years later Bernheim prepared an extract from acetone-dried baker s yeast, which had lactate dehydrogenase activity (5). Bach and co-workers demonstrated that the lactate dehydrogenase activity was associated with a 6-type cytochrome, which they named cytochrome 62 (6). In 1954, the enzyme was crystallized, enabling the preparation of pure material and the identification of flavin mononucleotide as a second prosthetic group (2). Since then, significant advances have been made in the analysis of the structure and function of the enzyme. Much of the earlier work on flavocytochrome 62 has already been summarized in previous review articles (7-10). In this article we shall describe recent developments in the study of this enzyme, ranging fi om kinetic, spectroscopic, and structural data to the impact of recombinant DNA technology. 

Xanthine oxidase, which is capable of catalyzing the conversion of hypoxanthine and xanthine to uric acid, was first detected in 1882 by Horbaczewski (Hll), who noted that extracts of various tissues could catalyze the conversion of xanthine to uric acid. A similar enzyme was detected in milk (M15). These enzymes contain a flavin-adenine dinucleotide prosthetic group (C9). As a result of the essential nature of the flavin-adenine dinucleotide portion of the enzjmie, a striking parallelism was seen between the riboflavin content of the diet and the xanthine oxidase concentration in tissues of growing rats (DIO). The enzyme contains molybdenum. That the molybdenum is contained in a functionally important component has been demonstrated by several workers (G13, T5). Totter and his associates injected labeled molybdate into a cow, and then isolated the enzyme from the milk to show that the proportion between the molybdenum and flavin remained constant at a value of 0.5. Corran et al. (C9) postulated that the xanthine oxidase of milk is identical with the xanthine oxidase of liver, but the protein portions of the enzyme appear to differ. 

The existence of the a and b forms of this enzyme has been known for many years contrary to an earlier conclusion, the b form preponderates in extracts of resting muscle. The o form (molecular weight 4.9 X 10 ) is converted into the b form (molecular weight 2.4 X 10 ) by PR enzyme, formerly regarded as a prosthetic-group-removing enzyme, later... 

When a protein possesses a prosthetic group such as heme, its concentration is usually determined at the absorption wavelengths of the heme. The most important absorption band of heme is called the Soret band and it is localized around 408-425 nm. The position of the peak of the Soret band depends on the heme structure, and in cytochromes, it will depend on whether the cytochrome is oxidized or reduced. For example, cytochrome c extracted from the yeast Hansenula anomala shows a Soret... 

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